Staining Intracellular Antigens for Flow Cytometry
抗胰岛细胞抗体检测试剂盒(化学发光免疫分析法)产品技术要求新产业
产品名称抗胰岛细胞抗体检测试剂盒（化学发光免疫分析法）型号、规格100测试/盒；50测试/盒结构及组成磁性微球：包被ICA抗原的磁性微球，PBS 缓冲液，牛血清白蛋白，防腐剂：叠氮钠；低/高点校准品：ICA，Tris缓冲液，鼠IgG，羊IgG，牛血清白蛋白，防腐剂：叠氮钠；发光标记物：ABEI 标记的抗人IgG，Tris缓冲液，鼠IgG，羊IgG，牛血清白蛋白，防腐剂：叠氮钠；缓冲液：牛血清白蛋白，防腐剂：叠氮钠；质控品1/2：ICA，Tris 缓冲液，鼠IgG，羊IgG，牛血清白蛋白，防腐剂：叠氮钠。
产品适用范围/预期用途本试剂用于体外定性检测人血清样本中抗胰岛细胞抗体，临床上主要用于I型糖尿病的辅助诊断。
2.性能指标2.1外观和性状抗胰岛细胞抗体检测试剂盒（化学发光免疫分析法）试剂盒各组分应齐全、完整、液体无渗漏；包装标签应清晰、准确、牢固；试剂盒内组分（磁性微球除外）应为澄清的液体，无沉淀、无悬浮物、无絮状物。
磁性微球悬浮溶液应该均匀分布，无肉眼可观察到的团聚颗粒，无异物，无块状沉淀。
第1 页共8 页2.2批内精密度检测抗胰岛细胞抗体企业参考品中精密度参考品，批内变异系数（CV）应≤8%。
2.3批间精密度检测抗胰岛细胞抗体企业参考品中精密度参考品，批间变异系数（CV）应≤15%。
2.4准确性（阳性符合率）检测抗胰岛细胞抗体企业参考品中阳性参考品，不得出现阴性反应（10/10）。
2.5特异性（阴性符合率）检测抗胰岛细胞抗体企业参考品中阴性参考品，不得出现阳性反应（15/15）。
2.6最低检出限检测抗胰岛细胞抗体企业参考品中最低检出量参考品，EL1~EL3 应检出阳性，EL4 应检出阴性。
2.7校准品2.7.1校准品准确度相对偏差应在±10%范围内。
2.7.2校准品瓶内重复性校准品瓶内重复性（CV）应≤8%。
2.7.3校准品均一性）应≤5%。
校准品均一性（CV均一性2.8质控品预期结果质控品1 每次测定结果应在（14.8~27.4）U/mL 范围内，质控品2 每次测定结果应在（36.3~67.3）U/mL 范围内。
人血管内皮细胞生长因子(VEGF)定量检测试剂盒(ELISA)
本试剂盒只能用于科学研究，不得用于医学诊断。
人血管内皮细胞生长因子（VEGF）定量检测试剂盒（ELISA）使用说明书【试剂盒名称】人血管内皮细胞生长因子（VEGF）定量检测试剂盒（ELISA）【试剂盒用途】定量检测人子血清、血浆及相关液体样本中血管内皮细胞生长因子（VEGF）的含量。
【检测原理】本试剂盒采用双抗体两步夹心酶联免疫吸附法（ELISA）。
将标准品、待测样本加入到预先包被人血管内皮细胞生长因子（VEGF））多克隆抗体透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分，再加入酶标工作液，温育足够时间后，洗涤除去未结合的成分。
依次加入底物A、B，底物（TMB）在辣根过氧化物酶（HRP）催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样品中人血管内皮细胞生长因子（VEGF））浓度呈正相关，450nm波长下测定OD值，根据标准品和样品的OD值，计算样本中人血管内皮细胞生长因子（VEGF））含量。
【试剂盒组成】1 酶标包被板12孔×8条7 底物夜A6mL2 标准品：1600pg/ml0.6mL 8 底物夜B6mL3 20倍浓缩洗涤液20mL9 终止液6mL4 标准品稀释液6mL10 说明书1份5 样本稀释液6mL 11 封板膜1张6 酶标试剂6mL12 密封袋1个备注：标准品用标准品稀释液依次稀释为：1600、800、400、200、100、50pg/ml【需要而未提供的试剂和器材】1、37℃恒温箱2、标准规格酶标仪3、精密移液器及一次性吸头4、蒸馏水5、一次性试管6、吸水纸【操作步骤】1、准备：从冰箱取出试剂盒，室温复温平衡30分钟。
2、配液：用蒸馏水将20倍浓缩洗涤液稀释成原倍的洗涤液。
3、加标准品和待测样本：取足够数量的酶标包被板，固定于框架上，分别设置标准品孔、待测样本孔和空白对照孔，记录各孔位置，在标准品孔中加入标准品50μL；待测样本孔中先加入待测样本10μL，再加样本稀释液40μL（即样本稀释5倍）；空白对照孔不加。
BD细胞固定液
BD Cytofix™Technical Data SheetFixation BufferProduct InformationMaterial Number:554655Size: 100 mlDescriptionBD Cytofix™ Fixation Buffer is comprised of a neutral pH-buffered saline (i.e., Dulbecco's Phosphate-Buffered Saline) that contains 4% w/v paraformaldehyde. This fixation buffer is intended to preserve human and rodent lymphoid cells for the subsequent immunofluorescentstaining of intracellular cytokines. BD Cytofix can also be used to preserve the light-scattering characteristics and fluorescence intensities ofhuman and rodent hematopoietic cells that have been stained by immunofluorescence for subsequent flow cytometric analysis.Preparation and StorageStore at 4° C and protected from prolonged exposure to light.Application NotesRecommended Assay Procedure:BD Cytofix can be used to fix unstained cells for subsequent immunofluorescent staining of intracellular cytokines. The suitability of fixing cellsfor immunofluorescent staining depends on whether the fluorescent antibodies can specifically detect their cognate antigens in a fixed form. Withrespect to intracellular cytokines, Pharmingen offers a large panel of conjugated anti-cytokine antibodies that can be successfully used to stainfixed and permeabilized cells. For the staining of antigens expressed on the surface of fixed cells, several fluorescent antibodies directed againstmouse cell surface antigens have been identified to be useful.BD Cytofix can also be used to fix cells after immunofluorescent staining in order to preserve the light-scattering signals and fluorescentintensities of cells for analysis at a later time. Cell Fixation Buffer may be useful to avoid the capping or shedding of fluorescent antibodies and/orsurface antigens during the period before flow cytometric analysis.Procedure for fixing cells with BD Cytofix™:1. Pellet 10e6 suspended cells (e.g., cytokine-producing cells generated by stimulatory culture) by centrifugation (250 - 300 x g) and carefullyremove supernatants to avoid cell loss.2. Add either 200 µl (for microwell plates) or 500 µl (for tubes) aliquots of cold DPBS containing protein and NaN3, gently resuspend cells,pellet, and remove supernatants.3. Repeat step 2.4. Add either 100 µl (for microwell plates) or 250 µl (for tubes) aliquots of fixation buffer to each cell pellet and resuspend the cells by eitherpipetting or vortexing. Incubate the cells with fixation buffer for 15 to 30 min at 4°C. (Cell aggregation can be avoided by vortexing prior to theaddition of the fixation buffer.)5. Fixed cells should be washed and suspended in a buffer that contains protein and NaN3, e.g., either Stain Buffer (FCS) [Cat. No. 554656] orStain Buffer (BSA) [Cat. No. 554657]. Store the fixed cells at 4°C (protected from light) for subsequent immunofluorescent staining ofintracellular cytokines. It is recommended that fixed cell samples be read as soon as possible, i.e., within one week.For the immunofluorescent staining of intracellular cytokines, cells that have been previously fixed with BD Cytofix™ can be washed two timesin a buffer that contains protein and NaN3 followed by incubating the cells for at least 10 minutes (4°C) in a buffer containing thecell-permeabilizing agent, saponin. BD Perm/Wash™ buffer (Cat. No. 554723) is ideally suited for this purpose. The fixed and permeabilizedcells can then be stained for intracellular cytokines as described in detail in the Immune Function handbook (BD Biosciences. 2003. Techniquesfor Immune Function Analysis, Application Handbook 1st Edition), available:/pdfs/manuals/02-8100055-21A1rr.pdf.Procedure for fixing immunofluorescently-stained cells with BD Cytofix™:Cells stained by immunofluorescence for cell surface antigens can be fixed as described above and stored (4°C, protected from light) forsubsequent analysis by flow cytometry (or fluorescence microscopy).NOTE : BD Cytofix/Cytoperm™ solution (Cat. No. 554722) and the BD Perm/Wash™ buffer (Cat. No. 554723) are included in BDCytofix/Cytoperm Kit (Cat. No. 554714) as well as the BD Cytofix/Cytoperm Plus Kit with GolgiStop™ (containing monensin; Cat. No. 554715)and BD Cytofix/Cytoperm Plus Kit with GolgiPlug™ (containing brefeldin A; Cat. No. 555028).Warnings and Precautions: BD Cytofix Buffer contains formaldehyde.R40Limited evidence of a carcinogenic effect.R43May cause sensitization by skin contact.S2Keep out of reach of children.S13 Keep away from food, drink and animal feedingstuffs.S23Do not breathe gas/fumes/vapour/spray.S36/37Wear suitable protective clothing and gloves.S46If swallowed, seek medical advice immediately and show this container or label.S56Dispose of this material and its container at hazardous or special wasteSuggested Companion ProductsCatalog Number Size CloneName554656Stain Buffer (FBS)500 ml(none) 554657Stain Buffer (BSA)500 ml(none) 554723Perm/Wash Buffer250 tests(none) 554714BD Cytofix/Cytoperm Fixation/Permeablization Kit250 tests(none) Product Notices1.Since applications vary, each investigator should titrate the reagent to obtain optimal results.2.Please refer to /pharmingen/protocols for technical protocols.ReferencesAlaverdi N, Waters JB. Pharmingen's Hotlines. 1997:6-15.(Methodology)BD Biosciences. Techniques for Immune Function Analysis, Application Handbook 1st Edition. 2003; Available:/pdfs/manuals/02-8100055-21A1rr.pdf 2007, Jan. 25.(Methodology)Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J Immunol Methods. 1981; 47(1):25-30.(Methodology)Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev. 1991;119:65-93.(Methodology)。
免疫学名词解释
1、Lymphocyte homing(淋巴细胞归巢):The process by which particular subsets of lymphocytes selectively enter and residue in peripheral lymphoid organs. It’s mediated by homing receptor on T lymphocytes and addressin on endothelial cells.2、Lymphocyte recirculation(淋巴细胞再循环):is the process by which lymphocytes continuously move between sites throughout the body through blood and lymphatic vessels, and it’s critical for the initiation and effectors phase of immune response.3、Hapten(半抗原):Substances which can combine with TCR, BCR or Ab, but cannot induce immune response independently.4、Sequential/Linear determinants(顺序型/线性决定簇):are epitopes formed by several adjacent amino acid residues. They exist on the surface or inside of antigen molecules. They are mainly recognized by T cells, but some also can be recognized by B cells.5、Conformational determinants(构象型决定簇):are epitopes formed by amino acid residues that aren’t ina sequence but become spatially juxtaposed in the folded protein. They normally exist on the surface of antigen molecules and can be recognized by B cells or Ab.6、T cell epitope(T细胞表位)：Antigen epitopes recognized by T cells(TCR).Features: peptides; sequential determinants; exist in anywhere of Ag; after processed, presented by MHC molecules; 8～23 amino acid residues long.7、TI-Ag/Thymus independent Ag(胸腺依赖性抗原): Ag which can stimulate B cells to produce Ab without the help of T cells and macrophages.8、TD-Ag/Thymus independent Ag(非胸腺依赖性抗原): Ag which can stimulate B cells to produce Ab with the help of T cells.9、Heterophile Ag/Forssman Ag(异嗜抗原): Common Ags shared by different species. They have no specificity of species and play an important role in immunopathology and diagnosis.10、CDR/Complementary determining region(互补决定区): The six HVR of H chain and L chain are brought together to form antigen-binding surface, so HVR is also called CDR.11、Idiotype/Idiotype determinant(独特型/独特型表位): The unique structure located in the V region of Ig ，BCR or TCR which show different immunogenicity due to the different amino acid sequence of HVR or CDR. It’s a unique Ag-specific marker for each Ig in an individual.12、Opsonization(调理作用):refers to the functions of IgG and IgM that after their Fab fragments bind Ag, their Fc fragments bind macrophage to enhance the phagocytosis of macrophage；or the functions of C3b and C4b on the microbe surface that bind to CR1 on the macrophage surface to enhance the phagocytosis of macrophage.13、ADCC/Antibody dependent cell mediated cytoxicity(抗体依赖的细胞介导的毒性作用):It’s a process in which FcR of NK cells, macrophages and monocytes bind to Fc fragment of Ab(IgG,IgA,IgE) coated target cells resulting in lyses of target cells.14、mAb /McAb/Monoclonal Ab (单克隆抗体):Ab produced by single B cell clone or one hybridomas clone and having a single specificity.(Immunized spleen cells(B) hybride with myeloma cells----hybridomas) 15、MAC/Membrane attack complex(攻膜复合体):C5b6789 complex which leads to formation of a hole in the membrane and mediates lysis of target cell.16、CKs/Cytokines(细胞因子):are small polypeptides produced in response to microbes and other Ags secreted by activated immunocytes or some matrix cells that mediate and regulate immune and inflammatory reactions by binding corresponding receptor on cells.17、IFN(干扰素):The CKs produced by human or animal tissue cells or T lymphocytes and NK cells,following the infection of virus and exposure to antigen whose principle function is to inhibit virusreplication or activate macrophage in both innate immunity and adaptive immunity.18、CAMs /Ams/cell adhesion molecules (黏附分子):The cell surface proteins involved in the interaction of cell-cell or cell-extracellular matrix. They play a crucial role in cell interaction, recognition, activation and migration by binding of receptor and ligand.19、CD/cluster of differentiation (分化簇):It is a group of cell surface molecules associated with the development and differentiation of immune cells.20、MHC/major histocompatibility complex(主要组织相容性复合体):A large cluster of linked genes located in some chromosomes of humanity or other mammals that encode major histocompatibility antigen and relate to allograft rejection, immune response and cell-cell recognition.21、HLA/Human leukocyte antigen(人类白细胞抗原):The major histocompatibility antigens for humanity which are associated with histocompatibility and immune response. They are alloantigens which are specific for each individual.22、HLA complex(HLA复合体):The MHC of humanity, a cluster of genes which encode for HLA and related to histocompatibility and immune response.23、MHC restriction(MHC 限制性):In interaction of T cell and APC or target cells, T cells not only recognize specific antigen but also recognize polymorphic residues of MHC molecules.24、PAMP/pathogen associated molecular pattern( 病原相关分子模式): The distinct structures or components that are common for many pathogens ，such as LPS, dsRNA of viruses etc.25、PRR/ pattern recognition receptor (模式识别受体): The receptors on macrophage that can recognize and bind PAMP on some pathogen, injured or apoptotic cells, including mannose receptor, scavenger receptor , toll like receptor etc.26、APC/Antigen presenting cells/Accessory cells/A cells(抗原递呈细胞): A group of cells which can uptake and process antigen and present antigen-MHC-Ⅰ/Ⅱcomplex to T cells, playing an important role in immune response.27、Cross-priming/Cross-presentation (交叉递呈): A mechanism by which a professional APC activates, a naïve CD8 CTL specific for the antigens of a third cell （e.g. a virus-infected or tumor cell）28、ITAM /immunoreceptor tyrosine-based activation motif(免疫受体酪氨酸活化基序): ITAM transduces activation signals from TCR, composing of tyrosine residues separated by around 18 aas. When TCR specially bind to antigen, the tyrosine becomes phosphorylated by the receptor associated tyrosine kinases to transduct active signals.29、TCR complex(TCR复合物): A group of membrane molecules on T cells that can specifically bind to antigen and pass an activation signal into the cell, consisting of TCR（αβ,γδ），CD3 （γε,δε）andδ-δ。
APC及抗原递呈(英文)
Cell surface
Exogenous antigens
Fusion Late endosome
Early endosome
Antigen degradation Golgi complex
MHC class 2 molecule Endoplasmic reticulum Ii chain
Processing and presentation of exogenous antigens
DC are the most powerful APC in vivo, which can effectively stimulate naï T cells to ve proliferate. Yet M and B cells can only stimulate activated or memory T cells.
(1) Production of endogenous antigenic peptide Ubiquitination of endogenous antigens Degradation of endogenous antigens in proteosome Peptides containing 8-12 amino acid residues
Professional APC
Dendritic cells, mononuclear phagocyte system, B cells, and APC that constitutively express MHC II molecules Non-professional APC
Dendritic cells
1 Antigen uptaking
DC capture antigens ：
干细胞活性因子让诗丹尼奥逆时动能原液逆转时光
干细胞活性因子让诗丹尼奥逆时动能原液逆转时光近几年，在护肤行业内常见到干细胞护肤这个概念，事实上干细胞护肤早已不是一个概念了，国内不少朋友已经享受到了干细胞护肤带来的好处。
在我国，干细胞护肤这一方面做得比较好的主要还是国健菁华（深圳）精准健康科技有限公司的旗下品牌诗丹尼奥。
毕竟他们软、硬件设施都比较完备，并且已经在该领域钻研了三十余年。
诗丹尼奥皮肤工程实验室是以清华大学吴耀炯教授为首席科学家，并荟聚了中国中医科学院、新西兰坎特伯雷大学等27位教授、博士团队在内的高、精、尖人才，团队完成了多项科研，并将获得的研究成果运用到护肤领域上。
诗丹尼奥产品的价值所在就是“干细胞活性因子”，这个活性因子对于修复肌肤有着极大的作用，而它也是能够让肌肤实现“逆转时光”的重要角色。
除了在干细胞领域取得了伟大的研发成果，诗丹尼奥也革新了在提取干细胞活性因子需要用到的技术。
首先是无血清培养，无血清培养可除去动物血清等过敏源又不影响细胞的生长；其次是3D培养，3D培养模拟细胞在体内的生长环境，增加细胞间的相互交流，提高间充质干细胞的干性和分泌功能，产出更多活性更强大的细胞因子。
通过这两大培养技术，能够更快更好地分离出活性更好且无致敏源的细胞因子，为后期产品的研发生产提供了更好的原料。
诗丹尼奥利用干细胞活性因子研发了逆时动能原液（面部）和逆时修护原液（妊娠纹）这两大明星产品。
两款产品均由清华大学教授带领团队研发，并且有多重专利支持，主要有效成分均为人体胎盘干细胞活性因子提取物。
逆时修护原液（妊娠纹）是针对妊娠纹、肥胖纹等萎缩纹研发的修复产品，它从根源性排除过敏源，打造最佳人体生理浓度配比，可修复皮肤纤维断裂，重塑光滑迷人肌肤，再生、修复二合一。
逆时动能原液(面部)是针对面部修护的护肤产品，除了有人体胎盘干细胞活性因子提取物外，它还包含了100多种活性小分子，让肌肤重获新生，逆转时光。
除此之外它还具有美白淡斑、抗氧化抗衰老、除皱紧致、促进伤口愈合、红润肌肤等功效。
安瓶幻灯
iStelle-术后极速修复系列
安
瓶
美国·艾思黛尔
目录
1. 美国iStelle(艾思黛尔)公司介绍
2. 中国总代理—万润时代公司介绍 3. 安瓶主要成分--干细胞信息素的来源及作用机理 4. 微脂囊包裹技术 5. 安瓶的主要成分、适用范围及使用方法。 6. 五年大样本临床观察及效果对比图。 7. iStelle(艾思黛尔)安瓶与竞品的比较 8. iStelle(艾思黛尔)系列介绍 9. 各类证书 10.小结
常见问题解答
iStelle(艾思黛尔)安瓶容量怎么才1.7ml，量太少了吧？
答： iStelle(艾思黛尔)安瓶的核心成份为高浓度的细胞信息素及玻尿酸，
具备高活性，同时应用了最先进的微脂囊技术，具有很强的透皮吸收功效， 使用1—2瓶完全可使术后肌肤得到很好的修复，如需要抑制瘢痕形成。需要 使用3个月左右。
术后第一天
40岁
女性
当日同时操作：水光针、5D
童颜术、自体血清填充三项. 术后第一天
美国·艾思黛尔
安瓶--同时做微整形三项效果
术后第三天
女士，40岁 同时操作：水光针、5D童颜术、 自体血清填充三项; 术后第三天。 术后7天淡妆出镜，接受采访
美国·艾思黛尔
安瓶—对儿童皮肤挫伤的修复对比
小朋友， 4岁,意外摔伤， 使用iStelle安瓶，每6小时涂 抹创面一次 48h完全结痂， 第5天结痂脱落,皮肤基本恢复 第6天皮肤完全恢复，没有一点
疤克：只能使用在完全愈合的伤口上，起到淡化疤痕的作用； 愈肤宁：主要为几聚糖(Chitosan),为含氨多糖，干燥后可形 成透明保护薄膜。 金扶宁 ：主要成分重组人粒细胞巨噬细胞刺激因子，主要为消炎的功效，没有促 进伤口愈合及细胞再生的作用；
免疫荧光步骤
Immunocytochemistry and immunofluorescence protocolProcedure for staining of cell cultures using immunofluorescenceICC and IF protocol Preparing the slide1.Coat coverslips with polyethylineimine or poly-L-lysine for 1 h at room temperature.2.Rinse coverslips well with sterile H2O (three times 1 h each).3.Allow coverslips to dry completely and sterilize them under UV light for at least 4 h.4.Grow cells on glass coverslips or prepare cytospin or smear preparation.5.Rinse briefly in phosphate-buffered saline (PBS).For wash buffer we recommend 1x PBS 0.1% Tween 20.FixationThe cells may be fixed using one of two methods:1.Incubating the cells in 100% methanol (chilled at -20°C) at room temperature for 5mining 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperatureThe cells should be washed three times with ice-cold PBS.Antigen retrieval (optional step)Certain antibodies work best when cells are heated in antigen retrieval buffer. Checkthe product information for recommendations for each primary antibody being used.1.Preheat the antigen retrieval buffer (100 mM Tris, 5% [w/v] urea, pH 9.5) to 95°C.This can be done by heating the buffer in a coverglass staining jar which is placedin a water bath at 95°C.ing a small pair of broad-tipped forceps, place the coverslips carefully in theantigen retrieval buffer in the cover glass staining jar, making note of which side ofthe coverslips the cells are on.3.Heat the coverslips at 95°C for 10 min.24.Remove the coverslips from the antigen retrieval buffer and immerse them, withthe side containing the cells facing up, in PBS, in the 6-well tissue culture plates.5.Wash cells in PBS three times for 5 min.PermeabilizationIf the target protein is intracellular, it is very important to permeabilize the cells.Acetone fixed samples do not require permeabilization.1.Incubate the samples for 10 min with PBS containing either 0.1–0.25% Triton X-100(or 100 μM digitonin or 0.5% s aponin). Triton X-100 is the most popular detergent forimproving the penetration of the antibody. However, it is not appropriate formembrane-associated antigens since it destroys membranes.2.The optimal percentage of Triton X-100 should be determined for each protein ofinterest.3.Wash cells in PBS three times for 5 min.Blocking and immunostaining1.Incubate cells with 1% BSA, 22.52 mg/mL glycine in PBST (PBS+ 0.1% Tween 20) for30 min to block unspecific binding of the antibodies (alternative blocking solutionsare 1% gelatin or 10% serum from the species the secondary antibody was raisedin: see antibody datasheet for recommendations).2.Incubate cells in the diluted antibody in 1% BSA in PBST in a humidified chamberfor 1 h at room temperature or overnight at 4°C.3.Decant the solution and wash the cells three times in PBS, 5 min each wash.4.Incubate cells with the secondary antibody in 1% BSA for 1 h at room temperaturein the dark.5.Decant the secondary antibody solution and wash three times with PBS for 5 mineach in the dark.3ICC and IF protocol Multicolor immunostaining(optional step)To examine the co-distribution of two (or more) different antigens in the same sample,use a double immunofluorescence procedure. This can be performed eithersimultaneously (in a mixture) or sequentially (one antigen after another).Ensure you have antibodies for different species and their corresponding secondaryantibodies. For example, rabbit antibody against antigen A, mouse antibody againstantigen B. Alternatively, you can use directly conjugated primary antibodiesconjugated to different fluorophores.Simultaneous incubation1.Incubate cells with blocking solution for 30 min.2.Incubate cells with both primary antibodies in 1% BSA in PBST in a humidifiedchamber for 1 h at room temperature or overnight at 4°C.3.Decant the solution and wash the cells three times in PBS, 5 min each wash.4.Incubate cells with both secondary antibodies in 1% BSA for 1 h at roomtemperature in the dark.5.Decant the secondary antibody solution and wash three times with PBS for 5 minseach in the dark.Sequential incubation1.First blocking step: incubate cells with the first blocking solution (10% serum fromthe species that the secondary antibody was raised in) for 30 min at roomtemperature.2.Incubate cells with the first primary antibody in 1% BSA or 1% serum in PBST in ahumidified chamber for 1 h at room temperature or overnight at 4°C, 1% gelatinor 1% BSA.3.Decant the first primary antibody solution and wash the cells three times in PBS, 5min each wash.4.Incubate cells with first secondary antibody in 1% BSA in PBST for 1 h at roomtemperature in the dark.5.Decant the first secondary antibody solution and wash three times with PBS for 5min each in the dark.6.Second blocking step: incubate cells with the second serum, (10% serum from thespecies that the secondary antibody was raised in) for 30 min at roomtemperature in the dark.47.Incubate cells with the second primary antibody in 1% BSA or 1% serum in PBST in ahumidified chamber in the dark for 1 h at room temperature, or overnight at 4°C.8.Decant the second primary antibody solution and wash the cells three times inPBS, 5 min each wash in the dark.9.Incubate cells with second secondary antibody in 1% BSA for 1 h at roomtemperature in the dark.10.Decant the second secondary antibody solution and wash three times with PBSfor 5 min each in the dark.If you have to detect more than two antigens, continue following steps 1–5 for therest of the antibodies.Counter staining1.Incubate cells with 0.1–1 μg/mL Hoechst stain or DAPI (DNA stain) for 1 min.2.Rinse with PBS.Mounting1.Mount coverslip with a drop of mounting medium.2.Seal coverslip with nail polish to prevent drying and movement under microscope.3.Store in dark at -20°C or +4°C.5。
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A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at the single-cell level. In this protocol, cells are first stained for surface antigens as in the surface antigen staining protocol, then fixed with formaldehyde to stabilize the cell membrane and permeabilized with the detergent saponin to allow anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in resting cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-γ, TNFα, IL-2, and IL-4, a combination of PMA (a phorbol ester, protein kinase C activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies can be used. To induce IL-6, IL-10 or TNF-α production by monocytes, stimulation with lipopolysaccharide (LPS) can be used. Note: The optimal stimulation condition for induction of a given cytokine is variable and must be determined empirically. For example, the best time for detection of IL-6-producing cells by human LPS-activated monocytes is 6 hours, whereas detection of IL-10 requires stimulation for at least 24 hours. In contrast to detection of secreted cytokines by ELISA, it is necessary to block secretion of cytokines with protein transport inhibitors, such as Monensin or Brefeldin A, during the last few hours of the stimulation for detection of intracellular cytokines by flow cytometry. It is advised that investigators evaluate the use and efficacy of different protein transport inhibitors in their specific assay system. The fixation and permeabilization buffers used for intracellular staining can have varying effects. eBioscience antibodies are optimized for use with the Foxp3 Staining Buffer Set (eBioscience Cat. No. 00-5523) or IC Fixation Buffer (eBioscience Cat. No. 00-8222) and Permeabilization Buffer (10X) (eBioscience Cat. No. 00-8333). Please contact Technical support for more information.
General Notes
1. 2. 3. 4. 5. For optimal performance of fluorochrome-conjugated antibodies, store vials at 4°C in the dark. Do not freeze. Prior to use, quick spin the antibody vial to recover the maximum volume. We do not recommend vortexing the antibody vial. Except where noted in the protocol, all staining should be done on ice or at 4°C with minimal exposure to light. Modifications relevant to staining with eFluor® nanocrystal (NC) reagents are noted in the general protocol by bold print. (Protocol B is recommended for better signal intensity) The fixation and permeabilization steps that are required for detection of intracellular antigens alter the light scatter properties and may increase non-specific background staining. Including extra protein such as BSA or FCS in the staining buffer can help reduce the non-specific background.
Protocol A: Two-step protocol for intracellular (cytoplasmic) proteins
The following protocol allows the simultaneous analysis of cell surface molecules and intracellular antigens at the single-cell level. In this protocol, fixation is followed by permeabilization. This results in the creation of pores in the cellular membrane that require the continuous presence of the permeabilization buffer during all subsequent steps to allow antibodies to have access to the cytoplasm of the cell. This mandates that all intracellular staining be done in the presence of the permeabilization buffer. This protocol is recommended for detecting cytoplasmic proteins, cytokines or other secreted proteins in individual cells following activation in vitro or in vivo. For cytokine detection, the appropriate stimulation conditions and kinetics of cytokine production will vary depending on the cell type and the particular cytokine being assayed. For in vitro stimulation of cells, it is necessary to block secretion of cytokines with protein transport inhibitors, such as the Monensin Solution (eBioscience Cat. No. 004505) or Brefeldin A Solution (eBioscience Cat. No. 00-4506), during the final hours of the stimulation protocol. For the detection of nuclear proteins such as transcription factors, please see Protocol B below. Materials