The dye-exclusion test for cell viability
流式细胞术检测单增李斯特菌与酿酒酵母_黄生权
流式细胞术检测单增李斯特菌与酿酒酵母黄生权1，付萌1，唐青涛1，黄韵2，胡双芳2，余以刚2，肖性龙2（1.无限极（中国）有限公司，广东江门 529156）（2.华南理工大学轻工与食品学院，广东广州 510640）摘要：为探讨流式细胞术对单增李斯特菌和酿酒酵母的活菌与热灭活菌的检出效果，本文采用荧光染色试剂SYTO-9和碘化乙锭（PI）对单增李斯特菌和酿酒酵母的活菌与热灭活菌的细胞悬液进行染色，采用流式细胞仪同时测量红色荧光与绿色荧光从而得出细胞悬液中的细菌和酵母的含量。
结果表明经核酸荧光染料染色后，再结合流式细胞术对细菌与酵母菌进行检测，步骤简单、耗时短。
该法不仅简化了测量步骤且分辨率高，对单增李斯特菌和酿酒酵母均具有良好的检出结果，能分辨同一体系中同一菌种的活细胞与热灭活细胞和同一体系中的细菌与酵母活细胞；该法检出限低，将单增李斯特菌稀释后，最低检出限可达1.2×104 cells/mL，将酿酒酵母稀释后，最低检出限可达6×103 cells/mL，因此能大大缩短增菌时间或者避免繁复的增菌步骤。
关键词：流式细胞术；荧光；李斯特菌；酿酒酵母文章篇号：1673-9078(2014)3-195-200Detection of Saccharomyces cerevisiae and Listeria monoeytogenes byFlow CytometryHUANG Sheng-quan1, FU Meng1, TANG Qing-tao1, HUANG Yun2, HU Shuang-fang2, YU Yi-gang2,XIAO Xing-long 2(1.Infinitus (China) Co. Ltd., Jiangmen 529156, China)(2.College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China)Abstract: The viable and heat-treated microorganisms such as Listeria monocytogenes and Saccharomyces cerevisiae were investigated in this study. They were stained by fluorescent staining reagents SYTO-9 and Propidium Iodide (PI) and then the red and green fluorescence signals were detected using flow cytometry to get the concentration of cells in samples. The results showed that flow cytometry was able to detect bacteria and yeast after the nucleic acid fluorescent staining. This method simplified the procedure for L. monocytogenes and S. cerevisiae detection, shortened the time of detection procedures, and also distinguished viable with heat-treated micrograms and viable bacteria with yeast in the same system. This method was capable of detecting as few as 1.2×104 cells/mL L. monocytogenes and 6 × 103 cells/mL S. cerevisiae. This improvement might shorten the time consuming of culture enrichment or simplify the process.Key words:flow cytometry; fluorescent; Listeria; Saccharomyces cerevisiae由于保健品原料中污染的微生物往往含量很低，或在加工过程中受到损伤活性低，给快速检测带来许多困难，由此造成的漏检给保健品生产企业带来重大的经济损失与资源浪费[1]。
大肠埃希菌产超广谱β-内酰胺酶的耐药性分析
.论著.大肠埃希菌产超广谱内酰胺酶的耐药性分析郭琼杰，杨柳，杨晨，王娜，王珊珊［摘要］目的分析大肠埃希菌(Escherichia coli,E.coli)产超广谱B-内酰胺酶(extended spectrum B-lactamases, ESBLs)的耐药性。
方法选取2017年6月〜2019年6月秦皇岛市第一医院收治的非重复大肠埃希菌感染80例患者作为研究对象，分别对其感染标本中的细菌进行检验、分离培养，运用常规鉴定技术鉴定细菌并进行药敏试验，使用纸片扩散法(K-B paper diffusion method,K-B法)检测E.coli,使用K-B法和国家临床实验室标准委员会(national committee for clinical laboratory standards,NCCLS)建议的双纸片协同试验进行ESBLs检测，统计E.coli ESBLs阳性株数并分析其耐药性。
结果非重复大肠埃希菌感染病例80例患者中E.coli ESBLs阳性株23株，占比28.75%,阴性株57例，占比71.25%。
E.coli ESBLs阳性株主要来源于尿液标本，之后依次为穿刺液、痰液、血液及引流液。
对E.coli ESBLs阳性株进行耐药性分析，结果显示亚胺培南的体外活性最好，耐药率最低为0.00%,其次为阿米卡星4.35%、头孢替坦4.35%、厄他培南4.35%、左氧氟沙星4.35%、头孢他啶4.35%、哌拉西林/他唑巴坦4.35%、妥布霉素4.35%；该试验中细菌对复方新诺明的耐药性最高为95.65%,其次为阿莫西林/克拉维酸91.30%、氨节西林91.30%、氨曲南86.96%、头孢曲松86.96%。
结论E.coli ESBLs对复方新诺明、阿莫西林/克拉维酸、氨节西林、氨曲南、头孢曲松有着较强的耐药性，对亚胺培南、阿米卡星、头孢替坦、厄他培南、左氧氟沙星、头孢他啶、哌拉西林/他唑巴坦、妥布霉素等药物较为敏感，临床治疗时应严格根据细菌分布特点结合其耐药情况进行合理给药。
Zombie Green Cell Viability Procedure
Zombie Green Viability Procedure:Materials and Equipment Needed:•Zombie Green Fixable Viabilty Kit (BioLegend, Cat# 423111 or 423112)•DPBS without Ca++ and Mg++•Cell Washing Buffer (DPBS+0.5% BSA+0.1%BSA) or BioLegend Cell Staining Buffer (Cat.# 420201) •Centrifuge with rotor and swing buckets•Flow cytometer with FITC detector•12 x 75 mm flow cytometry tubesProcedure:•Prepare Zombie Green Dye fresh daily as directed in the package insert. Follow all manufacturer’s instructions for storage of the kit and the prepared reagent.If processing PBMC, after first wash re-suspend cells in 10.5 mL to have sufficient volume.1.Pipet 500 µL of well-mixed cell suspension into a 12 x 75 mm tube.2.Add 2 mL of DPBS to wash.3.Centrifuge 300 x g for 5 min at RT.4.Decant.5.Re-suspend cells in 200 µL of DPBS.6.Add 2 µL of prepared Zombie Green dye *7.Vortex gently.8.Incubate 20 min in the dark at RT.9.Wash with 2 mL Cell Wash Buffer .10.Centrifuge 300 x g, 5 min11.Decant.12.Re-suspend cells in 300 µL of Cell Wash Buffer13.Keep cell suspensions in the dark and acquire as soon as possible.14.Acquire 20,000 gated (minus debris) events*If the positive Zombie Green signal is too bright, titration of the reagent is recommended. Gating: see Figure1.View SSC vs. FSC and gate out low FSC events to eliminate debris.2.View the minus debris population in a SSC vs. Zombie Green (FITC detector) dot plot.3.Gate Zombie Green low expression events. (high expression events are dead)4.Record the frequency of live events.minus debris78.5050K100K150K200K250KFSC-ASpecimen_001_Wash_Stain_002.fcsUngatedLive cells90.7-103103104105 FITC-A :: Zombie GreenSpecimen_001_Wash_Stain_002.fcsminus debrisFigure: Zombie Green gating。
BD 细胞活力试剂盒 (349483) 说明书
Becton, Dickinson and Company BD Biosciences2350 Qume Drive San Jose, CA 95131 USA 5/201523-6755-03***************************For Research Use Only. Not for use in diagnostic or therapeutic procedures.••••••••••••Live and Dead Cell Discrimination BD™ Cell Viability Kit Catalog No.Number of tests 349483100 Tests 349480 with BD Liquid Counting Beads 100 Tests RESEARCH APPLICATIONS Studies of:•rapid counting of live/dead bacteria or other microbial cells 1-4 •efficacy of bacterial disinfectant 5 •viability of yeast during fermentation 6 •viability and concentration of cells in culture •viability and concentration of cells before staining for flow cytometric analysis •mammalian or microbial cells •viability and count of cells in bioreactors •viability of sperm for research studies 7,8 •viability in cell preparations containing debris DESCRIPTIONFlow cytometry provides a rapid and reliable method to quantify viable cells in eukaryotic and prokaryotic cell suspensions.1-3,7,8 The BD Cell Viability Kit offers an easy-to-use dye combination to distinguish live and dead cells for analysis by flow cytometry. The kit contains thiazole orange (TO) solution to stain all cells and propidium iodide (PI) to stain dead cells. BD Liquid Counting Beads is a liquid suspension of fluorescent beads. Add the beads to a flow sample to calculate absolute counts. The kit can be ordered with or without counting beads. The method provides a rapid alternative to manual microscopic methods.4-6Live cells have intact membranes and are impermeable to dyes such as PI, which leaks into cells with compromised membranes. TO is a permeant dye and enters all cells, live and dead, to varying degrees. The fluorescent signal from TO in viable cells allows their enumeration even when debris in the cell preparation contaminates a scatter gate around the cells. Thus the combination of these two dyes provides a rapid and reliable method for discriminating live and dead eukaryotic and prokaryotic cells, including peripheral blood mononuclear cells (PBMCs), mammalian cell lines, bacteria, and yeast.MATERIALS PROVIDED The kit contains 1 vial of 500 µL 42 µmol/L TO in dimethyl sulfoxide (DMSO) and 1vial of 500 µL 4.3mmol/L PI in water. The optional BD Liquid Counting Beads are supplied as 1 vial of 10 mL of fluorescent microspheres in buffer with 0.1% sodium azide.Material required but not providedRecommended staining buffer: Physiologic phosphate-buffered saline containing 0.2% Pluronic® F68 and 1mmol/L EDTA.23-6755-03Page 2NOTE For bacteria, 0.01% TWEEN® 20 can be substituted for the Pluronic F68. The staining buffer should be passed through a 0.22-µm filter.HANDLING AND STORAGEStore vials at 2°–8°C with the TO and PI stored in the desiccated container provided. Each reagent is stable for the period shown on the bottle label when stored as directed.WARNINGSAll biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 9,10 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves.METHODRecommended amounts of TO and PI depend on the cell type being stained.Dilute cultured cells or PBMCs at least 1:10 in staining buffer to an approximate concentration range of 5x 105 to 107 cells/mL. (See Material required but not provided for preparation method.) For other sample types, such as pharmaceutical, food or environmental samples, at least 100 organisms per mL need to be detected using flow cytometry. If necessary, samples can be brought into this range by an initial concentration step.Bacteria and yeast: Add 5.0 µL of each dye solution to 500 µL of cell suspension. The final staining concentrations are 420 nmol/L for TO and 43 µmol/L for PI. Vortex and incubate for at least 5 minutes at room temperature.Mammalian cells: Add 4.0 µL of TO and 2.0 µL of PI solution to 2 mL of cell suspension. The final staining concentrations are 84 nmol/L for TO and 4.3 µmol/L for PI. Vortex and incubate for 5 minutes at room temperature.Bull semen: Add 2.0 µL of each dye solution to 2 mL of cell suspension. The final staining concentrations are 42 nmol/L for TO and 4.3 µmol/L for PI. Vortex and incubate for 5minutes at room temperature.NOTE When using BD Liquid Counting Beads, allow the beads to come to room temperature. Prior to analysis, gently vortex the bead suspension for 30 seconds and add 50 µL to each tube. Use the reverse pipetting technique to pipette for better accuracy. Cap the tubes and gently vortex to mix. Acquisition and analysis Acquire prepared samples on a BD FACS™ brand flow cytometer equipped with 488-nm laser excitation and BD CellQuest™ software. Use an FSC threshold for mammalian cells (fluorescence threshold if also using BD Liquid Counting Beads), and an SSC threshold for microbial cells. Gate cells using scatter and FL2.4-6 TO fluoresces primarily in FL1 and FL2; PI fluoresces primarily in FL3. Therefore, the best discrimination of live and dead populations is on an FL1 vs FL3 plot.To determine the concentration of the cell populations, use the following equation:Representative dataFigure 1 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a 500-µL sample of E. coli stained with 5 µL of TO and 5 µL of PI. Regions are set around the live, injured, and dead bacterial populations; counting beads are not shown.# events in cell region # events in bead region -----------------------------------------------------------# beads/test*test volume ---------------------------------×dilution factor ×concentration of cell population =* This value is found on the vial of BD Liquid Counting Beads and can vary from lot to lot.Page 323-6755-03Figure 1FL1 vs FL3 dot plot, gated on E. coli by scatter Figure 2 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a 2-mL sample of PBMCs stained with 4 µL of TO and 2 µL of PI. Live cells are in R5; dead cells are in R3; and counting beads are in R4.Figure 2FL1 vs FL3 dot plot, gated on PBMCs by scatter Figure 3 shows results obtained by adding 50 µL of BD Liquid Counting Beads to a 2-mL sample of Raji cells stained with 4 µL of TO and 2 µL of PI. Live cells are in R5; dead cells are in R3; and counting beads are in R4.Figure 3FL1 vs FL3 dot plot, gated on Raji cells by scatter Figure 4 shows results obtained by staining a 2-mL sample of thawed bull semen with 2 µL of TO and 2 µL of PI. For staining, 20 µL of thawed semen was added to 2 mL of staining buffer. Live sperm are in R3, dead sperm are in R2, and injured sperm are between the two regions.R6R5R4live injured dead FL1-H F L 3-H R3R4R5live dead injured FL1-H F L 3-H beads FL1-H F L 3-H R3R4R5dead injured live beads23-6755-03Page 4Figure 4FL1 vs FL3 dot plot, gated on thawed bull semen by scatter LIMITATIONSDispose of stained samples, extra dye solution, and container according to local, state, and federal regulations.CHARACTERIZATION To ensure consistently high-quality reagents, each lot of reagent is tested for conformance with characteristics of a standard reagent. Representative flow cytometric data is included in this data sheet.WARRANTYUnless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products.THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER . BD DISCLAIMS HEREBY ALL OTHER WARRANTIES , EXPRESSED OR IMPLIED , INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT . BD ’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE . BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES , INCLUDING PERSONAL INJURY , OR ECONOMIC LOSS , CAUSED BY THE PRODUCT . REFERENCES1.Nebe-von-Caron G, Stephens PJ, Badley AR. Bacterial detection and differentiation by cytometry and fluorescent probes. Proc Royal Microbiol Society . 1999;34:321-327.2.Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA. Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Meth . 2000;42:97-114.3.Davey HM, Kell DB. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev . 1996;60:641-696.4.Alsharif R, Godfrey W . Bacterial detection and live/dead discrimination by flow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002.5.Alsharif R, Tapia M, Godfrey W , Wanalund J, Nagar M. Bacterial disinfectant efficacy using flow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002.6.Thornton R, Godfrey W , Gilmour L, Alsharif R. Evaluation of yeast viability and concentration during wine fermentation using flow cytometry. Microbial Cytometry Application Note. BD Biosciences, Immunocytometry Systems; San Jose, CA. 2002.7.Graham JK. Assessment of sperm quality: a flow cytometric approach. Anim Reprod Sci . 2001;68:239-247.8.Yamamoto T, Mori S, Yoneyama M, Imanishi M, Takeuchi M. Evaluation of rat sperm by flow cytometry: simultaneous analysis of sperm count and sperm viability. J Toxicol Sci . 1998;23:373-378.9.Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline — Third Edition . Wayne, PA: Clinical and Laboratory Standards Institute; 2005. CLSI document M29-A3.10.Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR . 1988;37:377-388.FL1-H F L 3-H live injured dead R2R3TRADEMARKS AND LICENSE INFORMATION Pluronic is a registered trademark of BASF Corporation.Tween is a registered trademark of Croda International PLCBD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2015 BDThis product is provided under a license agreement between Life Technologies Corporation and Becton, Dickinson and Company through its BD Biosciences business, to use the fluorescent beads in this product only as standards, controls and calibrators for: (a) absolute counting; (b) fluorescence intensity measurements; and (c) instrument setup procedures on flow cytometry instruments, for calibrating and performance testing of flow cytometric instruments and flow cytometric assays, and for internal research and development applications. The sale of this product is expressly conditioned on the buyer not using the fluorescent beads in this product (1) in manufacturing; (2) for therapeutic, diagnostic (except when specifically labeled for such use in connection with BD products), or prophylactic purposes; (3) to resell, even if sold for calibration or research purposes. For information on purchasing a license to this product for purposes other than described above, contact Life Technologies Corporation, Cell Analysis Business Unit, Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300. Fax: (541) 335-0354.Page 523-6755-03。
血脑屏障氧糖剥夺再灌体外模型小鼠脑内皮细胞bEnd.3中微小RNA-290b-3p的作用及机制研究
血脑屏障氧糖剥夺再灌体外模型小鼠脑内皮细胞bEnd.3中微小RNA-290b-3p的作用及机制研究张奕雯1a，凡子莲1a，李经伦2，熊兰1a，赵自胜1b，李超1a作者单位：1广元市第一人民医院，a神经内科，b放射科，四川广元628000；2西南医科大学附属医院神经内科，四川泸州646000摘要：目的探讨氧糖剥夺再灌（OGD/R）条件下，小鼠脑内皮细胞bEnd.3中微小RNA-290b-3p（miR-290b-3p）在体外血脑屏障模型的作用与机制。
方法培养小鼠脑内皮细胞bEnd.3，建立OGD/R模型与体外血脑屏障模型，Transwell法检测氧糖剥夺后体外血脑屏障模型通透性的变化；实时荧光定量聚合酶链式反应（real time-PCR，RT-PCR）检测OGD/R后miR-290b-3p表达水平；Targetscan网站预测miR-290b-3p与紧密连接蛋白5（Claudin-5）结合；荧光素酶报告实验验证miR-290b-3p与Claudin-5结合；转染bEnd.3细胞miR-290b-3p抑制剂，应用蛋白质印迹法（Western Blot）检测Claudin-5的表达，Transwell法检测抑制miR-290b-3p后氧糖剥夺后体外血脑屏障模型通透性变化。
结果OGD/R后体外血脑屏障破坏，1、2、3、4、6h荧光素异硫氰酸酯（FITC）-葡聚糖扩散速率分别为（0.04±0.00）、（0.04±0.01）、（0.05±0.01）、（0.07±0.01）、（0.12±0.01）pmol·mm-2·min-1，通透性增加；miR-290b-3p在OGD/R后1、2、3、4、6h表达量分别为（1.18±0.12）、（1.27±0.12）、（1.55±0.18）、（2.13±0.18）、（2.53±0.15），表达增加；miR-290b-3p与Claudin-5结合；抑制miR-290b-3p可以缓解OGD/R后Claudin-5的降低，减轻血脑屏障的损伤。
J Pharmacol Exp Ther-2010-Arai-206-13[1]
![J Pharmacol Exp Ther-2010-Arai-206-13[1]](https://img.taocdn.com/s3/m/4fbeac8ed0d233d4b14e6935.png)
Metformin,an Antidiabetic Agent,Suppresses the Production of Tumor Necrosis Factor and Tissue Factor by Inhibiting Early Growth Response Factor-1Expression in Human Monocytes in VitroMasatoku Arai,Mitsuhiro Uchiba,Hidefumi Komura,Yuichiro Mizuochi,Naoaki Harada,and Kenji OkajimaDepartments of Translational Medical Science Research (M.A.,N.H.,K.O.)and Anesthesiology and Medical Crisis Management (H.K.,Y.M.),Nagoya City University Graduate School of Medical Sciences,Nagoya,Japan;and Department of Blood Transfusion and Cell Therapy,Kumamoto University Hospital,Kumamoto,Japan (M.U.)Received December 24,2009;accepted March 31,2010ABSTRACTMetformin,an antidiabetic agent,has been shown to reduce atherothrombotic disease in diabetic patients independent of antihyperglycemic effect.Recent studies have demonstrated that metformin attenuates the proinflammatory responses in human vascular wall cells and macrophages.However,the detailed molecular mechanisms underlying these therapeutic effects remain unclear.In the present study,we investigated the effects of metformin on tumor necrosis factor (TNF)pro-duction and tissue factor (TF)expression in isolated human monocytes stimulated with lipopolysaccharide (LPS)or oxi-dized low-density lipoprotein (oxLDL).Metformin significantly inhibited both TNF production and TF expression in isolatedhuman monocytes stimulated with LPS or oxLDL.Metformin also significantly inhibited TNF and TF mRNA in human monocytes stimulated with LPS.Although metformin did not inhibit the activation of either nuclear factor-B or activator protein-1,it inhibited the expression of early growth re-sponse factor-1(Egr-1)and phosphorylation of extracellular signal-regulated protein kinase (ERK)1/2in monocytes stim-ulated with LPS or oxLDL.These results suggest that met-formin may attenuate the inflammatory responses,at least in part,by suppressing the production of both TNF and TF through the inhibition of the ERK1/2-Egr-1pathway in human monocytes.Metformin is one of the most commonly used antidiabetic agents.It improves insulin sensitivity in patients with type 2diabetes (Stumvoll et al.,1995).In addition,metformin re-duces the risk of atherothrombotic disease accompanied with diabetes independent of its antihyperglycemic effect (UK Prospective Diabetes Study Group,1998).It is now well accepted that atherosclerosis is not merely a lipid disorder,but also an inflammatory disease (Ross,1999;Libby,2002).Inflammatory cells,such as monocytes/macro-phages,play a critical role in the initiation,progression,and complication of atherosclerotic lesions (Ross,1999;Libby,2002).Various cytokines contribute to pathogenesis of inflamma-tion observed in atherosclerosis (Kleemann et al.,2008).Among these cytokines,tumor necrosis factor (TNF)plays an important role.TNF is shown to be present in atherosclerotic lesions but not in normal vessels in humans (Tipping and Hancock,1993).TNF is associated with the progression of atherosclerotic lesions by regulating the functions of vascular wall cells to promote adhesion,migration,growth,and acti-vation of leukocytes (Young et al.,2002),thus leading to the further accumulation of mononuclear phagocytes and ampli-fication of inflammation (Ross,1999;Libby,2002).TNF is also associated with complications of atherosclerosis by in-creasing the production of matrix metalloproteinases contrib-uting to the vulnerability of the fibrous cap (Young et al.,2002).The activation of coagulation also contributes to the patho-genesis of atherosclerosis (Ross,1999;Libby,2002).Tissue factor (TF)is an important factor initiating blood coagulationArticle,publication date,and citation information can be found at .doi:10.1124/jpet.109.164970.ABBREVIATIONS:TNF,tumor necrosis factor;AMPK,AMP-activated protein kinase;AP-1,activator protein-1;Egr-1,early growth response factor-1;ELISA,enzyme-linked immunosorbent assay;ERK,extracellular signal-regulated protein kinase;JNK,c-Jun N-terminal kinase;LPS,lipopolysaccharide;NF-B,nuclear factor-B;oxLDL,oxidized low-density lipoprotein;TF,tissue factor;RT-PCR,reverse transcription-poly-merase chain reaction;I 〉,inhibitor B.0022-3565/10/3341-206–213$20.00T HE J OURNAL OF P HARMACOLOGY AND E XPERIMENTAL T HERAPEUTICSVol.334,No.1Copyright ©2010by The American Society for Pharmacology and Experimental Therapeutics 164970/3596086JPET 334:206–213,2010Printed in U.S.A.206at NSTL on May 14, 2012Downloaded from(Edgington et al.,1991),which is expressed in atheroscleroticlesions in humans(Tipping et al.,1989;Wilcox et al.,1989). Current evidence suggests that the exposure of TF-positive monocyte-derived macrophages to blood on plaque disruption may trigger both intravascular thrombosis and a sudden progression of atherosclerotic lesions(Libby,2002). Although various causes contributing to the pathogenesis of inflammation observed in atherosclerosis have been iden-tified,oxidized low-density lipoprotein(oxLDL)in hypercho-lesterolemia and lipopolysaccharide(LPS)from infectious microorganisms are recognized as potentially important sources of chronic inflammation in the development of ath-erosclerotic lesions(Ross,1999;Libby,2002).In the present study,we examined whether metformin inhibits TNF production and TF expression in human mono-cytes stimulated with LPS or oxLDL and attempted to iden-tify the underlying molecular mechanisms.Materials and MethodsMaterials.Metformin(1,1-dimethylbiguanide)was a generous gift from Nippon Shinyaku Co.,Ltd.(Kyoto,Japan).LPS(Esche-richia coli,serotype055:B5)was purchased from Sigma-Aldrich(St. Louis,MO).oxLDL was purchased from Intracel Resources,LLC (Frederick,MD).Polyclonal rabbit antibodies against inhibitorB (I〉)␣,p38,phosphorylated p38(Thr180/Tyr182),c-Jun N-terminal kinase(JNK),phosphorylated JNK(Thr183/Tyr185),extracellular signal-regulated protein kinase(ERK)1/2,phosphorylated ERK1/2 (Thr202/Tyr204),early growth response factor-1(Egr-1),and caspase-3were purchased from Cell Signaling Technology(Danvers, MA).Antibodies against PU.1and-actin were obtained from Santa Cruz Biotechnology,Inc.(Santa Cruz,CA).All reagents used were of analytical grade.Monocyte Preparation and Incubation.Human peripheral blood mononuclear cells were isolated from buffy coats provided by the local Central Institute of Blood Transfusion or obtained from healthy volunteer blood donors by Ficoll-Hypaque density gradi-ent centrifugation on Lymphoprep(Axis-Shield poC AS,Oslo, Norway)and then were further fractionated as described previ-ously(Uchiba et al.,1997).This study was performed with the approval of the ethics committee of the Nagoya City University Graduate School of Medical Sciences,and blood donors enrolled for the study gave informed consent in accordance with the Dec-laration of Helsinki.To minimize any possible interindividual variation among blood donors,each experiment was conducted by using monocytes from two to four buffy coats and repeated three times by using independent mononuclear cell preparations from different donors.The mononuclear cells in plastic dishes with RPMI medium1640(Invitrogen,Carlsbad,CA)supplemented with1%calf serum(HyClone Laboratories,Logan,UT)were in-cubated for16h at37°C in a humidified5%CO2incubator. Lymphocytes were then removed from the adherent monocytes by repeated rinsing with serum-free RPMI medium1640.The result-ing cell preparations wereϾ90%monocytes,as determined by May-Giemsa staining.Cell viability wasϾ95%,as determined by a trypan blue dye exclusion test.Monocytes thus obtained were adjusted to an appropriate volume and cultured in RPMI medium 1640supplemented with1%calf serum at37°C in a humidified5%CO2incubator.Various concentrations of metformin were added to cells at2h before stimulation with LPS(100ng/ml)or oxLDL (8g/ml).After further incubation under the same culture condi-tions for the required period,the cell suspensions were centri-fuged.The resulting supernatant fractions were used to deter-mine the levels of TNF,and the sedimented mononuclear pellets were used for TF activity measurements.Measurement of TNF Level.Human monocytes(1ϫ106cells/ assay)were stimulated with LPS for6h or oxLDL for12h in the presence or absence of metformin.The concentrations of TNF inculture media were determined by using an enzyme-linked immu-nosorbent assay(ELISA)kit for human TNF(BioSource Interna-tional,Camarillo,CA).Measurement of TF Activity.Human monocytes(1ϫ106cells/ assay)were stimulated with either LPS or oxLDL for6h in the presence or absence of metformin.The TF activities on monocyteswere measured as described previously(Molor-Erdene et al.,2005).In brief,human monocytes(1ϫ106cells)were washed twice by a phosphate-buffered saline and then stored atϪ80°C until TF activitywas measured.After thawing,the cell pellets were sonicated for30sto scrap the monocyte pellets and then dissolved in100l of clottingbuffer(12mM sodium acetate,7mM diethylbarbitate,and130mM sodium chloride).Fifty microliters of the resuspended cells wasmixed with50l of citrated plasma,and the clotting times were measured after recalcification with50l of20mM CaCl2solution at37°C.The TF equivalents were determined by using a standardcurve obtained from rabbit brain thromboplastin(Neoplastin Plus;Roche Diagnostic,Mannheim,Germany).Detection of Specific Binding of p65,p50,and c-Fos to DNAby Enzyme-Linked Immunosorbent Assay.Human monocytes(1ϫ107cells/assay)were stimulated with either LPS or oxLDL for1h in the presence or absence of metformin.Nuclear extracts were prepared as described previously(Yuksel et al.,2003).The specific binding of p65,p50,and c-Fos to their DNA consensus oligonucleo-tides was evaluated in nuclear extracts by using ELISA-based assaykits(Trans AM;Active Motif Inc.,Carlsbad,CA)as described previ-ously(Molor-Erdene et al.,2005).RNA Isolation and Quantitative mRNA Analysis.RT-PCRassays were used to assess TNF and TF mRNA levels in culturedhuman monocytes.Total RNA was extracted from cultured human monocytes by using TRIzol reagent(Invitrogen)according to the manufacturer’s instruction.This procedure yielded5to10g oftotal RNA from5ϫ105cells of cultured human monocytes.RNA samples were diluted in RNase-free water and stored atϪ80°Cuntil they were used.Real-time PCR was performed by using theABI PRISM7700Sequence Detection System,TaqMan One-StepRT-PCR Master Mix Reagents Kit,and commercially available predesigned,gene-specific primers and FAM-labeled probe setsfor quantitative gene expression(TaqMan Gene Expression As-says;human TNF code Hs00174128_m1,human TF codeHs00175225_ml,and human glyceraldehyde3-phosphate dehy-drogenase code Hs99999905_m1;Applied Biosystems,Foster City, CA).All probes used in these experiments spanned an exon–intron boundary.TNF,TF,and glyceraldehyde3-phosphate dehydroge-nase mRNA were quantified by parallel estimation.The thermalcycler conditions were30-min hold at48°C and10-min hold at95°C,followed by40cycles of15s at95°C and1min at60°C.Western Blot Analysis.Human monocytes(2ϫ106cells/assay)were stimulated with LPS or oxLDL for various times in the presenceor absence of metformin(10M).Whole-cell lysates were collected as described previously(Komura et al.,2008).Samples containingequal amounts of protein were separated by using sodium dodecylsulfate-polyacrylamide gel electrophoresis,and after electrophoresis,the proteins were transferred onto a polyvinylidene difluoride mem-brane(Bio-Rad Laboratories,Hercules,CA).The membranes were incubated with appropriate antibodies at4°C overnight and then subsequently with horseradish peroxidase-conjugated secondary an-tibody for1h at room temperature.Specific proteins were visualizedby using an enhanced chemiluminescence system(GE Healthcare,Little Chalfont,Buckinghamshire,UK).The densitometric quantifi-cation of the bands was performed by using National Institutes ofHealth Image software(version1.61).Cell Viability.Isolated monocytes were stimulated with LPS oroxLDL in the presence or absence of metformin.The number of monocytes was counted at6h after stimulation with LPS or12hInhibition of Egr-1Expression by Metformin207at NSTL on May 14, 2012Downloaded fromafter stimulation with oxLDL.Cell viability was evaluated by using a trypan blue dye exclusion test (Uchiba et al.,1997).Assessment of Apoptosis.Isolated monocytes were stimulated with LPS for 6h or oxLDL for 12h in the presence or absence of metformin (10M).Assessing the apoptosis of monocytes was performed by a Western blot analysis probed with anticaspase-3antibody.Statistical Analysis.The values are distributed in parametric manner and expressed as means ϮS.D.of one experiment represen-tative of three separate experiments that gave similar results.Dif-ferences in TNF production,TF activity,and mRNA levels were assessed by using analysis of variance followed by Scheffe ´’s post hoc test.In densitometric analysis data of Western blotting,the differ-ence between baseline values (time 0)and subsequent values for each experiment was compared by using analysis of variance fol-lowed by Scheffe ´’s post hoc test,and the differences between the values with and without metformin treatment at each time point were compared by unpaired t test.These analyses were carried out with StatView 5.0software (SAS Institute,Cary,NC).Statistical significance was defined as a level of p Ͻ0.05.ResultsEffect of Metformin on the Production of TNF and TF in Isolated Human Monocytes Stimulated with LPS.We examined the effect of metformin on LPS-induced increases in TNF production and TF activities in isolated human monocytes.We previously demonstrated that TNF production and TF activities by monocytes began to increase at 2h in monocytes stimulated with LPS (100ng/ml),peak-ing at 6h after the stimulation (Molor-Erdene et al.,2005;Komura et al.,2008).Metformin significantly inhibited the increases in both TNF production (Fig.1A;p Ͻ0.05)and TF activities (Fig.1B;p Ͻ0.05)in isolated monocytes after LPS stimulation.Metformin also inhibited the LPS-induced in-creases in TNF and TF mRNA levels in isolated human monocytes (Fig.2;p Ͻ0.05).The cell viability assessed by trypan blue dye exclusion test and apoptosis analyzed by Western blotting for caspase-3ruled out the possibility that the observed effects were caused by cell death in the experi-mental condition (data not shown).Effect of Metformin on the Activation of Nuclear Factor-B and Activator Protein-1Pathways in Iso-lated Human Monocytes Stimulated with LPS.Both NF-B and AP-1were demonstrated to be important tran-scription factors promoting the gene expression of TNF and TF in monocytes stimulated with LPS (Mackman,1995;Hambleton et al.,1996;Guha et al.,2001).To deter-mine whether metformin inhibits LPS-induced activation of NF-B in monocytes,we examined the effect of met-formin on LPS-induced degradation of I B ␣in isolated human monocytes.Metformin did not inhibit LPS-induced degradation of I B ␣in monocytes (Fig.3A).In addition,metformin did not inhibit LPS-induced increases in the DNA-binding activities of p65and p50to their consensus oligonucleotides (data not shown).To determine whether metformin inhibits LPS-induced activation of AP-1,we examined the effect of metformin on LPS-induced phos-phorylation of p38and JNK in isolated human monocytes.Metformin did not inhibit LPS-induced phosphorylation of p38and JNK in monocytes (Fig.3B and C).In addition,metformin did not inhibit LPS-induced increases in DNA-binding activities of c-Fos to their consensus oligonucleo-tides (data not shown).Effect of Metformin on the Expression of Egr-1and Phosphorylation of ERK1/2in Isolated Human Mono-cytes Stimulated with LPS.Egr-1has been demonstrated to be an important transcription factor promoting the gene expression of TNF and TF in monocytes stimulated with LPS (Mackman,1995;Guha et al.,2001).To determine whether metformin inhibits LPS-induced activation of Egr-1in mono-cytes,we examined the effect of metformin on LPS-induced increases in the expression of Egr-1in isolated human mono-cytes.Intracellular levels of Egr-1increased after LPS stim-ulation,peaking at 60min after stimulation and decreasing thereafter (Fig.4A).Metformin significantly inhibited the increases of Egr-1expression after 60min in monocytes stimulated with LPS (Fig.4A;p Ͻ0.05).The activation of ERK1/2was shown to induce transcription of TNF and TF byFig.1.Effects of metformin on increases in TNF production and TF activities in isolated human monocytes stimulated with LPS.A,human monocytes were preincubated with various concentrations of metformin for 2h and then stimulated with LPS (100ng/ml).Six hours after the stimulation,the supernatants were collected and TNF levels were mea-sured by ELISA as described in Materials and Methods .Data presented are means ϮS.D.(n ϭ4)and are representative of three separate experiments that gave similar results.ء,p Ͻ0.01versus LPS (Ϫ)without metformin;†,p Ͻ0.05versus LPS (ϩ)without metformin.B,human monocytes were preincubated with various concentrations of metformin for 2h and then stimulated with LPS (100ng/ml).Six hours after the stimulation,mononuclear cell pellets were collected and TF activities were measured by using the clotting assay method as described in Ma-terials and Methods .Data presented are means ϮS.D.(n ϭ4)and are representative of three separate experiments that gave similar results.ء,p Ͻ0.01versus LPS (Ϫ)without metformin;†,p Ͻ0.05versus LPS (ϩ)without metformin.208Arai et al.at NSTL on May 14, 2012Downloaded fromincreasing the expression of Egr-1(Guha et al.,2001).To determine whether metformin inhibits LPS-induced activa-tion of ERK1/2,thereby suppressing Egr-1expression,we examined the effect of metformin on LPS-induced phosphor-ylation of ERK1/2in isolated human monocytes.Intracellu-lar levels of phosphorylated ERK1/2increased after LPS stimulation,peaking at30min and decreasing thereafter (Fig.4B).Metformin significantly inhibited LPS-induced phosphorylation of ERK1/2in monocytes at30min after stimulation(Fig.4B;pϽ0.05).Effect of Metformin on the Production of TNF and TF in Isolated Human Monocytes Stimulated with oxLDL.oxLDL has been reported to induce TNF production and TF activity in isolated human monocytes in vitro (Jovinge et al.,1996;Petit et al.,1999).The effect of met-formin on oxLDL-induced increases in TNF production and TF activities was examined in isolated human monocytes. Metformin(10M)significantly inhibited the increases in both TNF production(Fig.5A;pϽ0.05)and TF activities (Fig.5B;pϽ0.05)in isolated monocytes at12and6h after oxLDL(8g/ml)stimulation,respectively.The cell viability assessed by a trypan blue dye exclusion test and apoptosis analyzed by Western blotting for caspase-3ruled out the possibility that the observed effects were caused by cell death in the experimental conditions(data not shown).Effect of Metformin on the Activation of NF-B, AP-1,and ERK1/2-Egr-1Pathways in Isolated Human Monocytes Stimulated with oxLDL.We examined whether metformin inhibits oxLDL-induced activation of NF-B and AP-1in monocytes.Metformin did not inhibit degradation of IB␣,phosphorylation of p38and JNK,or DNA-binding activities of p65,p50,and c-Fos in isolated human monocytes stimulated with oxLDL(data not shown). To determine whether metformin inhibits oxLDL-in-duced activation of Egr-1in monocytes,we examined the effect of metformin on oxLDL-induced increases in the expression of Egr-1in isolated human monocytes.Intra-cellular levels of Egr-1increased after oxLDL stimulation, peaking at120min after stimulation and decreasing there-after(Fig.6A).Metformin significantly inhibited the in-creases of Egr-1expression at120min in monocytes stim-ulated with oxLDL(Fig.6A;pϽ0.05).To determine whether metformin inhibits oxLDL-induced activation of ERK1/2,thereby suppressing Egr-1expression,we exam-ined the effect of metformin on oxLDL-induced phosphor-ylation of ERK1/2in isolated human monocytes.Intracel-lular levels of phosphorylated ERK1/2increased after oxLDL stimulation,peaking at60min after stimulation and decreasing thereafter(Fig.6B).Metformin significantly inhib-ited oxLDL-induced phosphorylation of ERK1/2in monocytes at 60min after stimulation(Fig.6B;pϽ0.05).DiscussionIn the present study,we demonstrated that metformin inhibited the production of TNF and TF in isolated human monocytes stimulated with LPS or oxLDL.The monocytic production of TNF and TF is regulated by various transcriptional factors including NF-B,AP-1,and Egr-1(Mackman,1995;Jovinge et al.,1996;Guha et al., 2001).The activity of NF-B is regulated primarily via its sequestration in the cytosol by anchoring to inhibitor protein IB␣(Baldwin,1996).As shown in the present study,pre-treatment with metformin did not affect the degradation of IB␣or the increase in DNA-binding activity of p65and p50 induced by LPS or oxLDL.These results suggested that the inhibitory effect of metformin on the production of TNF and TF in LPS-or oxLDL-stimulated monocytes might not be mediated by the inhibition of NF-B pathway.AP-1is another important factor regulating the production of TNF and TF.The activation of p38and JNK by phosphor-ylation was shown to enhance the transcriptional activity of AP-1(Mackman,1995;Hambleton et al.,1996;Jovinge et al., 1996).In the present study,metformin did not inhibit LPS-or oxLDL-induced phosphorylation of JNK and p38or the increase in DNA-binding activity of c-Fos.These results sug-gest that the inhibitory effect of metformin on the production of TNF and TF in monocytes might not be caused by the inhibition of AP-1activation.Metformin,on the other hand,inhibited LPS-or oxLDL-induced increases in intracellular levels of Egr-1as shown inFig.2.Effects of metformin on increases in TNF and TF mRNA in isolated human monocytes stimulated with LPS.A,TNF mRNA levels in isolated human monocytes preincubated with or without metformin(10M)for2h were determined at1h after stimulation with LPS(100ng/ml).TNF mRNA levels in human monocytes were detected by quan-titative RT-PCR as described in Materials and Methods.Data presented are meansϮS.D.(nϭ3)and are representative of three separate experiments that gave similar results.ء,pϽ0.01versus LPS(Ϫ)without metformin;†,pϽ0.05versus LPS(ϩ)without metformin.B,TF mRNA levels in isolated human monocytes preincubated with or without met-formin(10M)for2h were determined at1h after stimulation with LPS (100ng/ml).TF mRNA levels in human monocytes were detected by quantitative RT-PCR as described in Materials and Methods.Data pre-sented are meansϮS.D.(nϭ3)and are representative of three separate experiments that gave similar results.ء,pϽ0.01versus LPS(Ϫ)without metformin;†,pϽ0.05versus LPS(ϩ)without metformin.Inhibition of Egr-1Expression by Metformin209at NSTL on May 14, 2012Downloaded fromthe present study.Egr-1is shown to be rapidly and tran-siently expressed in monocytes in response to LPS or oxLDL (Guha et al.,2001;Harja et al.,2004).Because Egr-1is critically involved in the production of TNF and TF by mono-cytes (Mackman,1995;Guha et al.,2001),metformin might inhibit the production of TNF and TF by inhibiting the acti-vation of Egr-1in monocytes stimulated with LPS or oxLDL.Egr-1gene expression is regulated by various transcriptional factors,including Elk-1and Sap-1a.Guha et al.(2001)re-ported that Elk-1,activated by phosphorylated-ERK1/2,plays an important role in the Egr-1expression induced by LPS.They also reported that an inhibitor of ERK1/2pathway reduces LPS-induced production of TNF and TF via inhibi-tion of Egr-1expression in monocytes.Because metformin inhibited LPS-or oxLDL-induced phosphorylation of ERK1/2in the present study,it is probable that metformin may inhibit the expression of Egr-1by inhibiting ERK1/2activa-tion,thereby suppressing the production of TNF and TF in monocytes stimulated with LPS or oxLDL.The precise mechanism by which metformin inhibits phosphorylation of ERK1/2in monocytes stimulated with LPS or oxLDL remains unclear at present.Metformin is known as a pharmacological activator of AMP-activated protein kinase (AMPK)in various cell types (Zou et al.,2004;Hattori et al.,2006).However,it still remains con-troversial whether metformin exhibits an anti-inflamma-tory effect through the activation of AMPK.Hattori et al.(2006)demonstrated that high concentrations of met-formin (Ͼ1mM)reduce TNF-induced NF-B activity through the activation of AMPK in human umbilical vein endothelial cells.Isoda et al.(2006)reported that met-formin reduces high-glucose induced proinflammatory sig-naling independent of AMPK activation in human saphe-nous vein endothelial cells.Another AMPK activator,5-aminoimidazole-4-carboxamide riboside,was shown to ex-hibit anti-inflammatory effects independent of the activation of AMPK in RAW264.7cells stimulated with LPS (Jhun et al.,2004;Kuo et al.,2008).These observations indicate that the inhibitory effects of metformin on the production of TNF and TF in LPS-or oxLDL-stimulated monocytes might be independent of its activation of AMPK.TNF plays a critical role in proinflammatory responses in the development of inflammation observed in the ath-erosclerotic lesions of the vasculature (Tipping and Han-cock,1993;Libby,2002).TNF can regulate the various functions of vascular wall cells.TNF induces expression of adhesion molecules such as vascular cell adhesion mole-cule-1,intercellular adhesion molecule-1,E-selectin,andFig.3.Effects of metformin on the deg-radation of I B ␣and increases in intra-cellular levels of phosphorylated p38and phosphorylated JNK in isolated human monocytes stimulated with LPS.Isolated human monocytes pretreated with or without metformin (10M)for 2h were stimulated with LPS (100ng/ml)for the indicated times.Intracellular levels of I B ␣(A),phosphorylated p38(p-p38)(B),and phosphorylated JNK (p-p54and p-p46)(C)in human monocytes were deter-mined by Western blot analysis.-Actin,nonphosphorylated p38(p38),and non-phosphorylated JNK (p54and p46)were detected as the loading control.The re-sults of a densitometric analysis are also shown.Data presented are means ϮS.D.of three samples in one experiment rep-resentative of three separate experiments that gave similar results.E ,LPS without metformin;F ,LPS with metformin.ء,p Ͻ0.01versus time 0.210Arai et al.at NSTL on May 14, 2012Downloaded fromP-selectin(Marui et al.,1993;Collins et al.,1995;Taka-hashi et al.,1996;Young et al.,2002),crucial to the re-cruitment of mononuclear leukocytes to endothelial cells. TNF also increases the production of monocyte chemoat-tractant protein-1and interleukin-8,which are capable of inducing the migration of mononuclear leukocytes into the intima(Rollins et al.,1990;Young et al.,2002).TNF aug-ments the production of macrophage colony-stimulating factor by vascular wall cells,which stimulates the transi-tion of monocytes to lipid-laden macrophages(foam cells). Consequently,it promotes the survival and growth of foam cells and increases production of cytokines and growth factors by foam cells(Libby,2002;Young et al.,2002). Therefore,TNF is involved in the accumulation of mono-nuclear phagocytes and amplification of inflammation of the lesions leading to the development of atherosclerosis. The degradation of extracellular matrix by matrix metal-loproteinases is thought to be important in plaque rupture (Young et al.,2002).TNF is shown to stimulate the expres-sion of various types of matrix metalloproteinases in en-dothelial cells and smooth muscle cells and macrophages (Young et al.,2002).Because the present study has shown that metformin inhibits TNF production in LPS-or oxLDL-stimulated human monocytes,it is possible that the inhi-bition of TNF production by metformin in monocytes mayFig.4.Effect of metformin on increases in intracellular levels of Egr-1 and phosphorylated ERK1/2in isolated human monocytes stimulated with LPS.A,isolated human monocytes pretreated with or without metformin(10M)for2h were stimulated with LPS(100ng/ml)for the indicated times.Intracellular levels of Egr-1in human monocytes were determined by Western blot analysis.PU.1was detected as the loading control.The results of a densitometric analysis are also shown.Data presented are meansϮS.D.of three samples in one experiment repre-sentative of three separate experiments that gave similar results.E,LPS without metformin;F,LPS with metformin.ء,pϽ0.01versus time0;†, pϽ0.05versus LPS without metformin.B,isolated human monocytes pretreated with or without metformin(10M)for2h were stimulated with LPS(100ng/ml)for the indicated times.Intracellular levels of phosphorylated ERK1/2(p-ERK1/2)in human monocytes were deter-mined by Western blot analysis.Nonphosphorylated ERK1/2(ERK1/2) was detected as the loading control.The results of a densitometric anal-ysis are also shown.Data presented are meansϮS.D.of three samples in one experiment representative of three separate experiments that gave similar results.E,LPS without metformin;F,LPS with metformin.ء,pϽ0.01versus time0;†,pϽ0.05versus LPS without metformin.Fig.5.Effects of metformin on increases in TNF production and TF activities in isolated human monocytes stimulated with oxLDL.A,human monocytes were preincubated with metformin(10M)for2h and then stimulated with oxLDL(8g/ml).Twelve hours after the stimulation,the supernatants were collected,and TNF levels were measured by ELISA as described in Materials and Methods.Data presented are meansϮS.D.(nϭ4)and are representative of three separate experiments that gave similar results.ء,pϽ0.01versusoxLDL(Ϫ)without metformin;†,pϽ0.05versus oxLDL(ϩ)without metformin.B,human monocytes were preincubated with metformin(10M)for2h and then stimulated with oxLDL(8g/ml).Six hoursafter the stimulation,mononuclear cell pellets were collected,and TF activities were measured by using the clotting assay method as de-scribed in Materials and Methods.Data presented are meansϮS.D.(nϭ4)and are representative of three separate experiments that gavesimilar results.ء,pϽ0.01versus oxLDL(Ϫ)without metformin;†,pϽ0.05versus oxLDL(ϩ)without metformin.Inhibition of Egr-1Expression by Metformin211at NSTL on May 14, 2012Downloaded from。
无菌技术操作小结1000字
【一】:无菌操作基本技术无菌操作基本技术1. 实验进行前，无菌室及无菌操作台(laminar flow) 以紫外灯照射30-60 分钟灭菌，以70 % ethanol 擦拭无菌操作抬面，并开启无菌操作台风扇运转10 分钟后，才开始实验操作。
每次操作只处理一株细胞株，且即使培养基相同亦不共享培养基，以避免失误混淆或细胞间污染。
实验完毕后，将实验物品带出工作台，以70 % ethanol 擦拭无菌操作抬面。
操作间隔应让无菌操作台运转10 分钟以上后，再进行下一个细胞株之操作。
2. 无菌操作工作区域应保持清洁及宽敞，必要物品，例如试管架、吸管吸取器或吸管盒等可以暂时放置，其它实验用品用完即应移出，以利于气流之流通。
实验用品以70 % ethanol 擦拭后才带入无菌操作台内。
实验操作应在抬面之中央无菌区域，勿在边缘之非无菌区域操作。
3. 小心取用无菌之实验物品，避免造成污染。
勿碰触吸管尖头部或是容器瓶口，亦不要在打开之容器正上方操作实验。
容器打开后，以手夹住瓶盖并握住瓶身，倾斜约45° 角取用，尽量勿将瓶盖盖口朝上放置桌面。
4. 工作人员应注意自身之安全，须穿戴实验衣及手套后才进行实验。
对于来自人类或是病毒感染之细胞株应特别小心操作，并选择适当等级之无菌操作台(至少Class II)。
操作过程中，应避免引起aerosol 之产生，小心毒性药品，例如DMSO 及TPA 等，并避免尖锐针头之伤害等。
5. 定期检测下列项目：CO2 钢瓶之CO2 压力CO2 培养箱之CO2 浓度、温度、及水盘是否有污染(水盘的水用无菌水，每周更换)。
无菌操作台内之airflow 压力，定期更换紫外线灯管及HEPA 过滤膜，预滤网(300 小时/预滤网，3000 小时/HEPA)。
6. 水槽可添加消毒剂(Zephrin 1:750)，定期更换水槽的水。
培养基1. 液体培养基贮存于4 oC 冰箱，避免光照，实验进行前放在37 oC 水槽中温热。
Nature Research Reporting Summary说明书
October 2018Corresponding author(s):Sinem K. Saka, Yu Wang, Peng YinLast updated by author(s):June 05, 2019Reporting SummaryNature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist .StatisticsFor all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.The exact sample size (n ) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedlyThe statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)For null hypothesis testing, the test statistic (e.g. F , t , r ) with confidence intervals, effect sizes, degrees of freedom and P value notedGive P values as exact values whenever suitable.For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settingsFor hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomesEstimates of effect sizes (e.g. Cohen's d , Pearson's r ), indicating how they were calculatedOur web collection on statistics for biologists contains articles on many of the points above.Software and codePolicy information about availability of computer codeData collection Commercial softwares licensed by microscopy companies were utilized: Zeiss Zen 2012 (for LSM 710), Leica LAS AF (for Leica SP5), ZeissZen 2.3 Pro Blue edition (for LZeiss Axio Observer Z1), Olympus VS-ASW (for Olympus VS120), PerkinElmer Phenochart (version 1.0.2) .Data analysis Open-source Python (3.6.5), TensorFlow (1.12.0), and Deep Learning packages have been utilized for machine learning-based nucleiidentification (the algorithm and code is available at https:///HMS-IDAC/UNet). We used Matlab (2017b) for watershed-based nuclear segmentation using the identified nuclear contours. Python 3.6 was used for the FWHM calculations, as well as plotting ofhistograms. We used MATLAB and the Image Processing Toolbox R2016a (The MathWorks, Inc., Natick, Massachusetts, United States)for quantifications in mouse retina sections and for Supplementary Fig. 4. We utilized Cell Profiler 3.1.5 for the quantifications of signalamplification in FFPE samples in Figure 2 and 3. FIJI (version 2.0.0-rc-69/1.52n) was utilized for ROI selections and format conversions.HMS OMERO (version 5.4.6.21) was used for viewing images and assembling figure panels.For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.DataPolicy information about availability of dataAll manuscripts must include a data availability statement . This statement should provide the following information, where applicable:- Accession codes, unique identifiers, or web links for publicly available datasets- A list of figures that have associated raw data- A description of any restrictions on data availabilityData and Software Availability: The data and essential custom scripts for image processing will be made available from the corresponding authors P.Y.(**************.edu),S.K.S.(***********************.edu),andY.W.(********************.edu)uponrequest.Thedeeplearningalgorithmandtestdataset for automated identification of nuclear contours in tonsil tissues is available on https:///HMS-IDAC/UNet . The MATLAB code for nuclear segmentation isOctober 2018available on: https:///HMS-IDAC/SABERProbMapSegmentation .Field-specific reportingPlease select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.Life sciencesBehavioural & social sciences Ecological, evolutionary & environmental sciencesFor a reference copy of the document with all sections, see /documents/nr-reporting-summary-flat.pdfLife sciences study design All studies must disclose on these points even when the disclosure is negative.Sample size Each FFPE experiment batch were performed on consecutive sections from the same source, each containing over 600,000 cells. Due to largenumber of single cells with tens of distinct germinal center morphologies being present in each section, ROIs from different parts of a wholesection was used for quantification of signal improvement for each condition (consecutive sections were used for all the conditions of onequantification experiment). Number of ROIs are noted in the respective figure legends. For quantifications in retina samples, due toconserved staining morphology and low sample-to-sample variability n = 6 z-stacks were acquired from at least 2 retina sections. ForSupplementary Fig. 4, minimum 5 z-stacks were acquired for each condition to collect images of 18-45 cells. Number of cells are reported in the graphs.Data exclusions Parts of the FFPE tissue sections were excluded from analysis due to automated imaging related aberrations (out-of-focus areas) or tissuepreparation aberrations (folding of the thin sections at the edges, or uneven thickness at the edge areas). For FWHM calculations inSupplementary Fig. 2, ROIs that yield lineplots with more than one automatically detected peak were discarded to avoid deviations due tomultiple peaks. For Supplementary Fig. 4 cells in the samples were excluded when an external bright fluorescent particle (dust speck, dye aggregate etc.) coincided with the nuclei (as confirmed by manual inspection of the images). The exclusion criteria were pre-established.Replication Each FFPE experiment batch were performed on consecutive sections from the same source, each containing over 600,000 cells. Forevaluation and quantification of our method, multiple biological replicates were not accumulated in order to avoid the error that would beintroduced by the natural biological and preparation variation, and to avoid unnecessary use of human tissue material. In the case of themouse retina quantifications a minimum of two distinct retinal sections were imaged, and each experiment was performed at least twice. ForSupplementary Fig. 4 dataset, 16 different conditions were prepared and each were imaged multiple times (before linear, after linear, beforebranch, after branch). Although the data was not pooled together for the statistics reported in the figure, low cell-to-cell variability was observed and high consistency was seen across the samples for comparable conditions, suggesting low sample to sample variability.Randomization Randomization was not necessary for this study.Blinding Blinding was not possible as experimental conditions were mostly evident from the image data.Reporting for specific materials, systems and methodsWe require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.AntibodiesAntibodies used The full list is also available in Supplementary Information, Supplementary Table 4.Ki-67 Cell Signaling #9129, clone: D3B5 (formulated in PBS, Lot: 2), diluted 1:100-1:250 after conjugationCD8a Cell Signaling #85336 clone: D8A8Y (formulated in PBS, Lot: 4) diluted 1:150 after conjugationPD-1 Cell Signaling #43248, clone: EH33 (formulated in PBS, Lot: 2), diluted 1:150 after conjugationIgA Jackson ImmunoResearch #109-005-011 (Lot: 134868), diluted 1:150 after conjugationCD3e Cell Signaling #85061 clone: D7A6E(TM) XP(R) (formulated in PBS, Lot:2), diluted 1:150 after conjugationIgM Jackson ImmunoResearch #709-006-073 (Lot: 133627), diluted 1:150 after conjugationLamin B Santa Cruz sc-6216 clone:C-20, (Lot: E1115), diluted 1:100Alpha-Tubulin ThermoFisher #MA1-80017 (multiple lots), diluted 1:50 after conjugationCone arrestin Millipore #AB15282 (Lot: 2712407), diluted 1:100 after conjugationGFAP ThermoFisher #13-0300 (Lot: rh241999), diluted 1:50 after conjugationSV2 HybridomaBank, Antibody Registry ID: AB_2315387, in house production, diluted 1:25 after conjugationPKCα Novus #NB600-201, diluted 1:50 after conjugationCollagen IV Novus #NB120-6586, diluted 1:50 after conjugationRhodopsin EnCor Bio #MCA-A531, diluted 1:50 after conjugationCalbindin EnCor Bio #MCA-5A9, diluted 1:25 after conjugationVimentin Cell Signaling #5741S, diluted 1:50 after conjugationCalretinin EnCor Bio #MCA3G9, diluted 1:50 after conjugationVLP1 EnCor Bio #MCA-2D11, diluted 1:25 after conjugationBassoon Enzo ADI-VAM-#PS003, diluted 1:500Homer1b/c ThermoFisher #PA5-21487, diluted 1:250SupplementaryAnti-rabbit IgG (to detect Ki-67 and Homer1b/c indirectly) Jackson ImmunoResearch # 711-005-152 (Multiple lots), 1:90 afterconjugationAnti-mouse IgG (to detect Bassoon indirectly) Jackson ImmunoResearch #715-005-151) (Multiple lots), diluted 1:100 afterconjugationAnti-goat IgG (to detect Lamin B indirectly) Jackson ImmunoResearch # 705-005-147) (Lot: 125860), diluted 1:75 afterconjugationAlternative antibodies used to validate colocalization of VLP1 and Calretinin in Supplementary Fig. 8d-f:Calretinin (SantaCruz #SC-365956; EnCor Bio #CPCA-Calret; EnCor Bio #MCA-3G9 AP), VLP1 (EnCor Bio #RPCA-VLP1; EnCor Bio#CPCA-VLP1; EnCor Bio #MCA-2D11). All diluted 1:100.Fluorophore-conjugated secondary antibodies used for reference imaging:anti-rat-Alexa647 (ThermoFisher #A-21472, 1:200), anti-rabbit-Alexa488 (ThermoFisher #A-21206, 1:200), anti-rabbit-Atto488(Rockland #611-152-122S, Lot:33901, 1:500), anti-mouse-Alexa647 (ThermoFisher #A-31571, 1:400), anti-goat-Alexa647(ThermoFisher # A-21447, 1:200), anti-rabbit-Alexa647 (Jackson ImmunoResearch, 711-605-152, Lot: 125197, 1:300).Validation All antibodies used are from commercial sources as described. Only antibodies that have been validated by the vendor with in vitro and in situ experiments (for IHC and IF, with images available on the websites) and/or heavily used by the community withpublication in several references were used. The validation and references for each are publicly available on the respectivevendor websites that can reached via the catalog numbers listed above. In our experiments, IF patterns matched the distributionof cell types these antibodies were expected to label based on the literature both before and after conjugation with DNA strands. Eukaryotic cell linesPolicy information about cell linesCell line source(s)BS-C-1 cells and HeLa cellsAuthentication Cell lines were not authenticated (not relevant for the experiment or results)Mycoplasma contamination Cell lines were not tested for mycoplasma contamination (not relevant for the experiment or results)Commonly misidentified lines (See ICLAC register)No commonly misidentified cell lines were used.October 2018Animals and other organismsPolicy information about studies involving animals; ARRIVE guidelines recommended for reporting animal researchLaboratory animals Wild-type CD1 mice (male and female) age P13 or P17 were used for retina harvest.Wild animals The study did not involve wild animals.Field-collected samples The study did not involve samples collected from the field.Ethics oversight All animal procedures were in accordance with the National Institute for Laboratory Animal Research Guide for the Care and Useof Laboratory Animals and approved by the Harvard Medical School Committee on Animal Care.Note that full information on the approval of the study protocol must also be provided in the manuscript.Human research participantsPolicy information about studies involving human research participantsPopulation characteristics We have only used exempt tissue sections for technical demonstration, since we do not derive any biological conclusions, thepopulation characteristics is not relevant for this methodological study.Recruitment Not relevant for this study.Ethics oversight Human specimens were retrieved from the archives of the Pathology Department of Beth Israel Deaconess Medical Centerunder the discarded/excess tissue protocol as approved in Institutional Review Board (IRB) Protocol #2017P000585. Informedinform consent was waived on the basis of minimal risk to participants (which is indirect and not based on prospectiveparticipation by patients).Note that full information on the approval of the study protocol must also be provided in the manuscript.October 2018。
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324
YIP AND AUERSPERG
TABLE 1 ])YES USED FOR VIABILITY ])ETERMINATIONS~ ARRANGED IN ASCENDING ORDER OF ~OLECULAt WEIGHTS
Dye (Source) Molecular Weight Concentration Reported for Viability Counts
MATERIALS AND METHODS
Dyes. Seven dyes, listed in Table 1, were studied. Six of these, i.e. safranin (8), eosin (7), congo red (9), erythrocin (7), trypan blue (2), and nigrosin (10), had been used previously for dye-exclusion tests. Alcian blue, a water-sohlble stain with a relatively high molecular weight (M.W. 1341), was added because preliminary * This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada. t Recipient of a Research Studentship from the National Cancer Institute of Canada.
work showed that, over brief periods of time, trypan blue (M.W. 961) but not safranin (M.W. 351) or eosin (M.W. 692) stained only a small proportion of glutaraldehyde-fixed cells, and this observation suggested a possible dependence of the differential staining oil the molecular weight of tile dye used. In order to compare the staining properties and the rate of diffusion into cells of these dyes, the), were made to "t uniform molal (m) concentration. The concentration that was selected (2.26 X 10-4 m) was the lowest one among those reported for viability determinations (Table 1) to minimize possible toxic effects of the dyes on living cells. Stock solutions (• 10) were made in Hanks' BSS except for alcian blue, which at this concentration precipitates in BSS or in phosphate buffer within a few hours. Therefore, a solution of 6.0% alcian blue in 95% ethanol was prepared and diluted with distilled water, 1:20, to obtain an aqueous stock solution (• of 0.3% alci'm blue and 4.5% ethanol. This solution was stable for several months. Cell culture. The following cell lines were used in the present study: (a) IRC (12), a strain of rat leukemia, cultured as stationary suspension cultures in Fischer's medium with 10% horse serum, 100 units per ml of penicillin, and 100/~g per inl of streptomycin; (b) KB 14, a human carcinoma line, cultured as a monolayer in Waymouth's medium MB 752/1 with 10% fetal calf serum, 100 units per ml of penicillin, and 100 tLg per nfl of streptomycin. Cell death or damage that resulted in ~taining by the dye-exclusion test was induced by either
SUMMARY
A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde fixation. The proportion of viable cells in a cell population can be estimated in various ways including methods based on the reduction of 2,3,5-triphenyltetrazolium chloride (1) vital staining and the uptake of labeled thymidine (2), but the simplest one and the one most widely used is the so-called "dye-exclusion" method (3), which depends on the phenomenon that many stains are excluded by living cells but not by dead cells (3-7). While the staining procedure is quite simple, it is difficult to process large numbers of samples concurrently, particularly where the ex'tct timing of progressive eytotoxie effects is required. It therefore seemed desirable to determine whether the differential staining of dead and live cells was based on structural differences that would persist following fixation, so that the dye-exclusion test could be performed on fixed cells at convenient times after the main experimental work. The present communication describes such a method.
g/liter equivalent molality, m ]
Concentration Equivalent to 2.26 X _ _-~ I0 m*
g/liter
Safranin (B.D.tt.) . . . . . . . . . . . . . . . . . . . . . Eosin Y (B.D.H.) . . . . . . . . . . . . . . . . . . . . . Congo red (Gurr) . . . . . . . . . . . . . . . . . . . . . . Erythrocin B (Fisher Sei. (2o.) . . . . . . . . . Trypan blue (B.D.H.) . . . . . . . . . . . . . . . . . Nigrosin (Allied Chem. (2o.) . . . . . . . . . . Alcian blue (Harleco) . . . . . . . . . . . . . . . . . 1972
THE DYE-EXCLUSION TEST FOR CELL VIABILITY: PERSISTENCE OF DIFFERENTIAL STAINING FOLLOWING FIXATION*
DOMINIC K. YIPt AND NELLY AUERSPERG Cancer Research Centre and Department o] Zoology, University o] British Columbia, Vancouver 8, Britislt Columbia, Canada