流式实用技巧-merckmillipore
merck millipore 默克密理博纯水超纯水一体系统明澈D24UV操作手册
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明澈-D 24 UV
需要的物品
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物品检查清单 安装开始前请核实所有物品是否可用。
o 纯水系统主机 (A) o 用户手册和快速参考指南 (B) o 外部电源 (C) o PROGARD®预处理柱和 Q-GARD®超纯水柱（D） o 储水水箱：30、60 或 100 升（E） o POU 取水器（F） o 包含下述的附件包： 管材 配件 电源
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2 显示器和小键盘
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3 Progard® 预处理柱位置
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4 锁定手柄
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5 消毒/清洁口
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6 电源接口
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7 系统进出水口连接
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*系统断电时，应将电源与插座断开。
说明 系统 ID 和序列号 电源适配器* Q-GARD®超纯水柱位置 超纯水进出口接口 POU（用水点）取水POU 取水开关取水器终端过滤器
重要提示！
应在干净且干燥的地方安装并操作您的纯水系统。请参考本手册最 后部分的环境要求。
您的纯水系统不适于家庭使用。
纯水系统长时间断电会使计时器的电池放电，导致失去时间和日期记录。如果长 时间关闭纯水系统，应使用实验室关闭模式请与您的服务代表联系。
经常保存系统的历史记录-每三个月或每半年。参见 Millitrack 文档。
软件选项millitrack软件是通过ajax异步javascript和xml技术开发的一种嵌入式gui图形用户界面软件它能运用tcpip以太网协议通过点到点计算机或直接通过网络连接生成交互式网页
用户手册
明澈™-D 24UV 纯水系统
序言
感谢您购买我们的纯水系统。 对于任何疑问或请求，请根据以下联系信息与 Merck Millipore 联系。
Merck Millipore 30L-100L水存储胶囊说明书
Storage Tanks and AccessoriesStorage with a DifferenceMerck Millipore is a business ofGuarantee the purity of your stored waterPure water requires a storage system to prevent the degradation of your water quality. Merck Millipore’s 30-, 60-, and 100-liter polyethylene (PE) storage tanks are designed to maintain consistent purity of stored water and provide effective protection against airborne contaminants.*Prevent contamination Water stagnancy can cause bacterial proliferation. Our optimal Automatic Sanitization Module (ASM) provides the ideal solution for the prevention of bacterial growth and biofilm formation on the inner surface of the storage tank. In addition, our advanced vent filter protects pure water from airborne contamination.Distribute your stored water where it is neededTo provide pure water for use with all of their applications, laboratories need to be able to distribute stored water from their water purification system storage tanks.- For distribution of non-pressurized pure water,conveniently located on the front of the Merck Milliporestorage tanks.- For convenient distribution the storage tank, an E-POD® point-of-delivery dispenser can be connected to an Elix® Advantage or Milli-Q® Integral water purification system.- For automatic feed of pure water, distribution valves on the base of the storage tank allow connection to other laboratory equipment such as glassware washing machines. Distributionpumps are also available if needed.* A complete line of storage tanks is available, ranging in capacity from a few liters to several hundred liters . Your nearest Merck Millipore office will be able to guide you in the choice of the tank best suited to your needs.4562Unique features• Polyethylene selected for its minimum release of extractables • Opaque walls block sunlight to prevent algae development • Smooth inner surface prevents biofilm formation• Cylindrical shape minimizes surface area in contact with water • Conical bottom allows complete draining for cleaning and rinsing • Pure water smoothly fed in at the bottom of the tank prevents absorption of carbon dioxide• Front valve enables manual dispense of pure water• Distribution valves permit connection to other laboratory equipment • Hermetically sealed lid blocks air from entering the tank• Large top opening allows manual cleaning during sanitization procedure • Compact space-saving designFail-safe protection• Sensor rod float switch system for automatic storage tank refill and indication of water level (% full)• Overflow connected to the drain, in the unlikely event of a water system malfunction• Direct display of stored water level on water purification system units • Water sensorMerck Millipore 30-, 60-, and 100-liter polyethylene storage tanks incorporate the latest technical developments and advanced features for stored water of consistent purity.All tanks have a small footprint and are designed for wall-mounting if required. Underbench installation is also possible for some models.For details of the tests performed during the storage tank development process, please request the publication “R &D Notebook 1: Optimizing the storage of purified water for laboratory applications” (Ref. No.: RD001EN00)from your local Merck Millipore representative.Optimized pure water storageInnovative storage tank designStorage tanks designed for efficiencyThe main concern when storing pure water is degradation of water purity over time. Only a strict choice of storage tank materials, associated with a careful design and appropriate protection against airborne contaminants, can ensure consistent water quality during storage.Designed for efficiency1. Hermetically sealed lid2. Sensor rod float switch3. Sanitary overflow4. Blow-molded storage tank5. Cylindrical shape6. Conical bottom with distribution valves7. Front dispensing valve8.Advanced vent filter3Advanced storage tank vent filterStorage Tank AccessoriesIn order to help ensure optimum purity and distribution of your stored water, Merck Millipore offers a range of accessories and connections for your storage tank, including the following items:Advanced vent filter:important protection against airborne contaminantsAir is contaminated by carbon dioxide, particles, microorganisms, and volatile organic compounds that come mainly from the laboratory atmosphere. To protect pure water from all these contaminants, Merck Millipore has developed an advanced storage tank vent filter that includes:• Activated carbon to adsorb volatile organics (including lab solvents such as acetone, chloroform, and methanol)• A soda-lime bed to remove CO 2• A Durapore® hydrophobic membrane for particle and bacteria retentionThis advanced vent filter is recommended for the protection of high-resistivity water, such as Elix® product water, during storage. To protect RiOs™ reverse osmosis-quality water, a Durapore® 0.45 µm hydrophobic membrane vent filter is also available.1. Airborne Contaminants A. Volatile Organics B. Particles C. Bacteria D. CO 22. Volatile Organics absorption3. CO 2 removal4. Particle and Bacterial retention5. Storage tank inletPurified air enters the storage tank• Advanced Vent Filter• Automatic Sanitization Module • Air Gap Device• E-POD® pure water remote dispenser • Washer Distribution Kit • Water Sensor • Lab Close KitComplete Merck Millipore water purification chain with the ASM and water sensor4Automatic Sanitization Module (ASM):say “No!” to bacterial proliferation Maintaining high purity water with low bacteria levels during storage is critical. If left to proliferate, trace levels of microorganisms present in pure water compromise water purity. This bacterial contamination is responsible for the formation of a biofilm — an accumulation of organic material made up of active and dead organisms, on the inner walls of the storage tank.Even though chemical sanitization and mechanical scrubbing may be periodically performed, this biofilm is difficult to remove and is a frequent source of recontamination in stored water.Merck Millipore’s ASM is designed to prevent the growth and proliferation of bacteria and the resulting biofilm on the inner surface of Merck Millipore PE storage tanks. The ASM makes use of the germicidal properties of an ultraviolet (UV) light at 254 nm, which is fitted inside the tank.The ASM provides full flexibility for guaranteed results• 254 nm UV lamp; selected for its germicidal effectiveness • Pre-programmed intervals of 10-min / day automatic UV illumination for optimized efficiency• Additional programmable and manual UV exposure possible to meet critical application requirements• Up to 45 min /day of UV exposure for total flexibility• Program daily time settings, UV cycles, and UV lamp operation displayed on the Millitrack® e-Solution dashboard • UV lamp exchange alarm for easy maintenance• Compact design allowing installation on top of the storage tank10 minutes of daily UV exposure is sufficientDuring development of the ASM, the UV lamp exposure cycles were optimized by examining the resulting bacterial reduction after exposure.Two 60-liter storage tanks were fed by an intentionally contaminated reverse osmosis water purification system. The tanks were then emptied and refilled each day and alternately equipped with an ASM into which variable illumination times were programmed.As shown in the graphs, 10 minutes per day of UV exposure were enough to make the tanks return to their original low bacterial levels.Biofilm formation over timeGermicidal effectiveness of the UV lamp5Air gap device for protection against bacteriological contaminationWater purification systems and storage tanks sometimes require a connection to the drain. Drains are typically dirty environments contaminated by microorganisms, and in particular, bacteria. Therefore, when the outlet of the water system reject tubing is pushed into the drain, there is a risk that bacteria could contaminate the inside of the reject tubing, and then progressively move to the water system.One way to prevent this from occurring is to install an air gap device on the reject tubing. This allows the reject water flow to movethrough the tubing without touching the inside of the contaminateddrain environment. Installing an air gap device is an easy and safe way to prevent the development of bacteria above the air gap level.The E-POD® Elix® water point-of-delivery unit can be connected to an Elix® Advantage pure water system or Milli-Q® Integral pure and ultrapure water system to dispense pure water wherever it is needed in the lab.Advantages of the E-POD® remote dispenser include: • Improved bacterial water quality(less than 0.1 cfu/mL, with final filter)• V ersatility enabling use for multiple applications or users when a Millipak® or Biopak® polisher is fitted to the dispenser outlet• Volumetric dispensing to save time • Ergonomic design and ease of use• Flexibility, with installation of up to three E-POD® units per system• Information at a glance thanks to the color backlit screen on the dispenser base •Space-saving small footprintE-POD® pure water remote dispenserAir Gap DeviceE-POD® pure water remote dispenser: pure water where you need it6Water sensor for control over feed water supplyIf there is water on the floor, the water sensor enables shutdown of the feed water supply in order to prevent a lab flood.Lab Close Kit keeps your system in top condition when you’re away When your facility is closed for an extended time — such as vacation periods — the Lab Close K it will avoid water purification system standstill during these long periods. The Lab Close K it intelligently optimizes the consumption of water and electricity by your system, preventing the negative effects of nonuse, such as bacteria buildup. Your water purification system remains in top condition, ready forimmediate use upon your return.Distribution pumps to meet increased demandsWasher Distribution KitA key use of pure water is as feed to laboratory appliances such as glassware washers, autoclaves, sterilizers and weathering devices.The Merck Millipore Washer Distribution Kit provides cost-effective and convenient distribution of pure water to common laboratory appliances, with flow rates between 12.5 – 13.5 L/min (at 2 bar or 30 psi, depending on voltage).Installation of the small-footprint kit is fast, easy, and universal, with bench, underbench, or wall-mounted options. Users profit from a silent, automatic supply of pure water when required.The graph shows characteristics of Merck Millipore distribution pumps.Pumps provide a long service lifetime and quiet operation.Washer Distribution KitSpecifications & Ordering InformationSpecifications for Polyethylene Storage TanksThere are several different storage tank sizes available in this range:* For 30-liter and 100-liter storage tanks, underbench models are also available.Ordering InformationPolyethylene Storage Tanks7For more information, please visit our website:/labwaterPB5506EN00 Copyright 2015 EMD Millipore Corporation, Billerica, MA., U.S.A.Merck Millipore, the M mark, Milli-Q, Elix, AFS, E-POD, Millitrack, Millipak, Biopak, and Durapore are registered trademarks of, and RiOs is a trademark of, Merck KGaA, Darmstadt, Germany.All rights reserved.。
流式细胞术最全攻略：从protocol到问题解决
流式细胞术最全攻略：从protocol到问题解决文献中我们经常能看见这种图，也就是流式结果分析图，美观、简单、一目了然。
流式细胞术（flow cytometry）是20世纪60年代后期开始发展起来的利用流式细胞仪快速定量分析细胞群的物理化学特征以及根据这些物理化学特征精确分选细胞的新技术，主要分为流式分析和分选2部分。
今天我们主要介绍流式分析中基本操作与技巧，首先简要了解一下什么是流式细胞术。
一、流式细胞术的3大要素1. 流式细胞仪：现在市面上有多种型号的流式细胞仪，但其基本结构都是相同的，分析性流式细胞仪有液流系统、光路系统、监测分析系统。
（1）液流系统（2）光路系统光路系统始于激光器，其分类分法很多，最常用的分类方法是根据其发射的激光的波长来分，如常见的有488nm的蓝激光器、635nm的红激光器和405nm的紫激光器，不同的激光器发出的激光照射到细胞后产生的光信号会经过不同的光路系统被不同的通道接收。
流式细胞仪采集的光信号包括散射光信号和荧光信号，散射光信号也就是我们常说的FSC（前向散射光，反应细胞的大小）、SSC（侧向散射光，反应细胞的复杂程度）；荧光信号是细胞上结合有荧光素，被激光激发以后，会发射荧光信号。
（3）检测分析系统：流式细胞仪的检测分析系统就是以通道为单位将细胞的各个通道的光信号汇总分析，最后得出样品群体中细胞的物理化学特征。
通道，我们可以理解为就是光电倍增管，有2个作用，①将光信号转变为电子信号；②放大电子信号。
可以分为散射光通道（FSC通道、SSC通道）和荧光通道，一个激光器下可以有多个荧光通道，一个通道对应多个荧光染料，例如以beckman的DXflex机器的638nm 红激光器为例，APC通道可以接收APC、Alexa Fluor647、eFluor660荧光信号，原则上，这3种荧光素不能同时标记一个样品，否则，就无法分析该通道上的信号是来自于哪种荧光素。
另一个重要组成部分就是计算机分析系统，例如BD的DIVA系统。
细胞活性的检测方法比较
细胞活性的检测方法比较简介：细胞活性是进行细胞生物学实验的一项重要指标，是评价细胞培养、细胞毒性及生理研究的基础。
一些基于细胞的下游分析实验，需要前期有好的细胞，才能保证下游的实验结果的可信度以及实验的可重复性，因此，细胞活性的实验具有广泛的应用。
目前关于细胞活性的检测方法多种多样，如基于台盼蓝染色的细胞计数，以及在此基础上的图像分析型的计数设备，或者是基于荧光染料标记的流式细胞术的分析等。
本文将一种新的检测方法与传统的检测方法进行比较，考察不同方法之间数据的差异性与方法学本身的操作性和成本进行比较。
细胞活性的定义具有多样性，通过对关键的细胞生理指标进行标记来进行检测，如细胞代谢的活性（酯酶的功能，MTT法）、凋亡标记物（Annexin V）、细胞氧化还原电位、膜电位、增值率（DNA含量）、线粒体功能和膜的完整性等。
虽然有如此多的指标参数，但是基于对细胞膜的完整性和相关的染料排斥发检测，已经被广泛的认为是作为细胞活性检测的标准。
应用最广泛的细胞膜完整性的检测方法是台盼蓝染色法，该法最早是基于显微镜下观测并且手工计数，过程费事费力，并且计数的系统误差较大。
为了排除人工计数的误差，增加计数过程的准确性，出现了基于仪器放大、拍照计数的方法。
这样提高了计数过程的自动化程度，但是依旧无法摆脱由于计数时焦平面的选择、颗粒识别的准确性问题，并且容易高估细胞活性。
为了解决这些问题，一些荧光染料被发明进行使用。
该法能够较台盼蓝染色提高100倍或者更高的检测准确性与灵敏度。
这类型的荧光染料中，碘化丙啶（Propidium Iodidi，PI）是应用最广泛的一种标准染料。
与其他基于荧光染料标记的实验一样，该法的缺点就是需要使用荧光显微镜或者是流式细胞仪。
前者要求配置一套荧光显微镜，相对应的激发光源以及滤光片，并且操作过程依旧是手工操作，人工计算实验结果，过程耗时还无法避免人为的主观因素。
而流式细胞仪能够准确的计数，并且进行统计学分析，但是设备昂贵，需要有经验的实验人员进行专门操作，以及需要一定的设备维护等问题。
流式细胞分析技术
ü 光电倍增管（PMT）：
检测散射光和荧光；同时将光学信号转换成 电脉 冲信号。
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滤光片
分色反光镜
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PMT 滤光片
荧光和散射光检 测系统
分色反光镜
激发光
激光聚光系统
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数据处理系统
• 计算机及其软件组成 • 进行实验数据的分析、存储、显示
Photons/Detector (V)
光信号转化为电脉冲信号
特点：单细胞、快速、高通量、多参数、准确、灵敏
经典流式细胞仪（分析型和分选型） 量化成像分析流式细胞仪 质谱流式细胞仪
单激发光；4个 荧光检测通道
经典流式细胞仪
双激发光；6个 荧光检测通道
Coulter EPICS XL
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量化成像分析流式细胞仪
美国merck millipore公司
质谱流式细胞仪
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经典流式细胞仪
液流系统-液流聚焦原理
喷 嘴
鞘液(Sheath）
喷嘴
荧光信号或 侧向散射光
样本流
前向散射光 单个细胞流
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液流系统形成单 个细胞流示意图
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光路系统
ü 激发光：
常用的是空冷式的氩离子激光光源（488nm）；氦氖 激光光源（633nm)；紫外光源
ü 各种光学镜片：
分色反光镜 光束成形器；透镜 滤光片：长通滤片；短通滤片；带通滤片
(一）标本采集、运输、保存和操作 l标本来源
l几乎所有组织细胞均可用于FCM检测 l免疫标本主要来源于外周血、骨髓、 淋巴器官或组织等
l抗凝剂选择
l肝素钠(首选) lEDTA-Na2
l标本保存
标本制备
ü 外周血或骨髓样本
流式细胞仪常用技术方法)
流式细胞仪常用技术方法细胞周期/DNA分析(PI法)一、鞘液准备(至少3L PBS,提前一天准备)0.01M的PBS配制方法:800ml蒸馏水中溶解NaCl8.0g;KCl0.2g;Na2HPO40.24g;调节pH到7.2-7.4(用HCl或NaOH调节,各地蒸馏水的酸碱度不同),加蒸馏水定容至1L,0.22um滤膜过滤后,室温保存。
二、制备一个阴性对照来设定流式细胞仪的取样参数和十字门的范围:没有染色的细胞(制备过程如下,仅不加抗体)三、实验步骤1.取对数期生长细胞,倒去培养液,胰酶适度消化细胞,用培养液吹打,1000rpm,离心10min弃上清;2.PBS洗2次,加0.5ml吹匀,务必吹散;3.加入70%预冷乙醇中(标准做法是将细胞悬液加入预冷70%酒精),固定时可加入Triton X-100以增加膜的通透性)封口膜封口,4℃固定1-2h或过夜(可长至一个月);4.1000rpm×10min离心收集细胞,PBS洗两次;5.用0.4mlPBS重悬细胞并转移至离心管中轻轻吹打;6.加入Rnase-A约3ul至终浓度50ug/ml,37℃水浴消化30min;7.加入PI约50ul至终浓度约为5-50ug/ml,室温避光染色30min;8.用300目滤网过滤,上机检测。
细胞周期/DNA分析(BD公司CycleTEST PLUS DNA Reagent Kit)一、鞘液准备(至少3L PBS,提前一天准备)0.01M的PBS配制方法:800ml蒸馏水中溶解NaCl8.0g;KCl0.2g;Na2HPO40.24g;调节pH到7.2-7.4(用HCl或NaOH调节,各地蒸馏水的酸碱度不同),加蒸馏水定容至1L,0.22um滤膜过滤后,室温保存。
二、制备一个阴性对照来设定流式细胞仪的取样参数和十字门的范围:没有染色的细胞(制备过程如下1-6)三、若做DNA倍体分析,需另设附加对照管肿瘤细胞和外周血单核细胞的混合管,比例至少为2:1四、实验步骤1. 将细胞消化后,用冷PBS洗细胞两次,300g×5min(若细胞数过少可提高转速到500g),为减少细胞损失可用1.5ml离心管离心;2. 弃上清留大约50ul下面的液体防止碰到细胞团,加入1ml Buffer Solution并低速混匀细胞。
流式细胞仪使用一般方法及技巧
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TITER
40
30 20
10
0
0
1
2
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Dilution
孵育、溶血、固定
• 抗体孵育：室温下避光15分钟（某些情况下如：细胞
内因子检测需冰育） • 溶血：如样本含中有红细胞需溶血（见下页） • 样本溶血后需立即上机检测，固定后可放置较长时间
白细胞保护型溶血剂Cal-Lyse(其中已含细胞固定剂)：
标本处理时间：
新鲜样本分析时，样本采集后应立即分析
样本固定方法：
1%的多聚甲醛或75%的酒精，作用30分钟。 注：不同的实验，所用的固定方法不完全相同。
样本处理标准试剂：
一、10%Bovine Serum Albumin BSA
1. 溶解10g BSA至100ml蒸馏水中 2. 4º C，20000 g离心30分钟
APC
• 激发光633nm， 发射光660 在配有双激光的流 式细胞仪上，此荧 光染料与FITC、PE、 PE-Cy5联用做四色 分析。但现在因单 激光四色可实现， 故使用该染料意义 不大。
核酸染料
染料 Propidium Iodide Ethidium biromide 7-Aminoactinomycin D Acridine Orange Chromomycin A3 Hoechst 33342 DAPI Pyronin Y Thiazole Orange YO-PRO-1 TO-PRO-3 LDS 751 激发光 发射光 495342 639 493320 637 546 647 503 530(DNA) 640(RNA) 430 580 395 450 372 456 545 565 509 533 642 661 642 661 543 712 光源 488 488 514488 488 457 UV UV 514488 488 488 630 488
Millisolve 清澈系统用户指南说明书
User GuideMillisolve™ Clarification System Cat. No. XX1614700IntroductionThe Millisolve™ Clarification System is designed for vacuum filtration of liquid chromatography (LC) buffers and solvents. The system uses a disposable 0.2/0.22 or 0.45 micron (µm) filter to remove particles which can shorten column life. Vacuum filtration through this system can also remove a large portion of dissolved gases from buffers, reducing the risk of air bubble interference with LC instrumentation.NOTE: To avoid solvent and buffer cross-contamination, EMD Millipore Corporation recommends a dedicated filtration system for each solvent and use of a new filter to process each batch of solvent.The Millisolve™ Clarification System is supplied with two different filter holder configurations. For hazardous fluids, the vacuum cap configuration allows filtration to take place in a closed system. When vacuum is applied, fluid is drawn directly from a bottle, through the filter, and into the conical Millisolve™ flask. For batch filtration, fluid is poured into the open 300 milliliter (mL) funnel and flows through the filter and into the Millisolve™ flask when vacuum is applied. The following table shows the types of membrane filters appropriate for filtering various solvents and buffers.Type of Solvent or Buffer Filter to Use Pore Size Cat. No.Water, buffers MF-Millipore™ hydrophilic mixed celluloseesters filter (Triton® surfactant-free)0.22 µm0.45 µmGSTF04700HATF04700Nylon hydrophilic membrane filter0.2 µm0.45 µm GNWP04700 HNWP04700Omnipore™ hydrophilic polytetrafluoroethylene (PTFE) filter0.2 µm0.45 µmJGWP04700JHWP04700LCR hydrophilic PTFE filter0.45 µm FHLC04700Water, buffers, alcohol, or mixtures Durapore® polyvinylidene fluoride (PVDF)hydrophilic filter0.22 µm0.45 µmGVWP04700HVLP04700 Nylon hydrophilic membrane filter0.2 µm0.45 µmGNWP04700HNWP04700 Omnipore™ hydrophilic PTFE filter0.2 µm0.45 µmJGWP04700JHWP04700 LCR hydrophilic PTFE filter0.45 µm FHLC04700All organic solvents (methylene chloride, hexane, tetrahydrofuran)Fluoropore™ hydrophobic PTFE filter0.2 µm0.45 µmFGLP04700FHLP04700orFHUP04700 Omnipore™ hydrophilic PTFE filter0.2 µm0.45 µmJGWP04700JHWP04700 LCR hydrophilic PTFE filter0.45 µm FHLC04700Additional Equipment Required●47 millimeter (mm) filter appropriate for your application●Vacuum source, either central laboratory vacuum or a portable vacuum pump. See Ordering Information foravailable models.Y WARNING: Choose a vacuum source appropriate for the fluid being filtered. Many vacuum pumps are NOT explosion-proof and should not be used when filtering flammable or explosive liquids.●Vacuum tubing, 3/16 inch (in.) (4.8 mm) inner diameter (ID) silicone rubber (cat. no. XX7100004)Millisolve™ Clarification System ComponentsVacuumNOTE: Smooth-tip filter forceps are supplied but not shown.A vacuum base with a stainless steel screen filter support (instead of a coarse-frit glass support) is also available(cat. no. XX1514732). The stainless steel screen filter support is easier to clean, and improves the filtration rate. PrecautionsY WARNING: Filtration of flammable liquids can generate enough electrostatic charge to cause a fire or explosion. To mitigate this potential, follow these precautions:●DO NOT use the stainless steel screen filter support with highly flammable solvents.●Work under a hood (or another ventilation system) when filtering flammable liquids toprevent accumulation of ignitable vapor and air mixtures. Ventilation systems, includingfume hoods, must be designed in accordance with the National Fire Protection Association(NFPA) code.●Keep the filtration rate low; apply only enough vacuum pressure to maintain steady flowthrough the filter.●Follow safe handling guidelines for laboratory glassware when working with the Millisolve™ Clarification System.Information on safe handling of glassware can be found at /labware/glassware.●Although this product is rated for 948 mbar (28 in. Hg), it is not necessary to apply maximum vacuum pressure toachieve a reasonable filtration rate. Whenever possible, operate at lower vacuum pressure in order minimize the potential for glassware breakage due to implosion.●Before use, soak new glassware in 1% hydrochloric or nitric acid for several hours, then wash and rinse thoroughly with laboratory grade water . This neutralizes the slight alkalinity of the new glass and ensures that loose particles (glass beads) are removed from the fritted glass filter support. Refer to the Cleaning section for details on cleaning after use. ●To protect the vacuum source from fluid contamination, install a vacuum trap flask between the Millisolve™ flask and the vacuum source and place a Millex ®-FA 50 filter (cat. no. SLFA05010) in the vacuum line between the vacuum trap flask and vacuum source.Millisolve™ system Vacuum trap Vacuum pump●When using the vacuum cap for continuous filtration, make sure that the filter is dry. If the filter is wet at the beginning of the filtration process (before the solvent/buffer flow enters the vacuum cap), there will be no flow because the vacuum pressure is not high enough to overcome the filter bubble point. If the filtration process is interrupted or air is introduced into the line, use a new, dry filter on a dry support to restart filtration.Operation1. Connect one end of the vacuum tubing to the sidearm (outlet) of the vacuum base. Push gently to secure thetubing, wetting the sidearm with water , if necessary.2. Optional : Stretch the funnel alignment guide over the bottom ofthe vacuum cap or funnel with the tab facing down and aligned with vacuum cap sidearm .If the alignment guide covers the sealing surface of the vacuum cap or funnel, it is upside down. Remove and change guide alignment guide orientation so that the sealing surfaces of the cap/funnel and base can be assembled in direct contact with one another .NOTE: The alignment guide is helpful in aligning the cap or funneland base, but it is not required.3. For continuous filtration using the vacuum cap , connect an appropriate length of PTFE tubing to thesidearm (inlet) of the cap.NOTE: Use the shortest length of tubing possible between the solvent container and the vacuum cap. Slowly push the tubing over the sidearm of the cap. If necessary, soften it first by immersing the end (approximately 1 centimeter [cm]) in hot water before pushing it over the sidearm.4. Place the Millisolve™ vacuum flask on a flat surface close to the vacuum source. Place the base on top of theflask.5. If using the stainless steel screen filter support, install it in the base, making sure that the supplied PTFE gas-ket is installed under the support.6. With smooth-tip forceps, center a 47 mm filter disc on thesupport surface.7. Without disturbing the filter, center the flange of the vacuum capor funnel on top of the base and lock the cap or funnel and basetogether with the spring clamp.8. Connect the free end of the vacuum tubing to the vacuum source.9. For continuous filtration (using the vacuum cap), pour the buffer/solvent to be filtered into a beaker,Erlenmeyer flask, or other suitable laboratory-grade container. Immerse the end of the PTFE tubing into the solutionto be filtered, making sure that the end reaches to the bottom of the container.For batch filtration (using the funnel), pour the solution to be filtered into the funnel.CAUTION: Make sure that the volume you want to filter does not exceed the 2 L capacity of the Millisolve™ flask.A 5 L flask (XX1614706) is available for larger volumes.10. Apply vacuum. Do not exceed 948 millibar (mbar) (28 in. Hg).For continuous filtration, the liquid will be drawn into the PTFE tubing, through the vacuum cap, through thefilter, and into the Millisolve™ flask.11. After all of the buffer/solvent has filtered, turn off the vacuum and disconnect the vacuum tubing from the vacuumsource.12. Remove the spring clamp, and lift the cap off of the base. Place the cap on a clean surface.13. With smooth-tip forceps, remove the filter from the base and discard.Using the Filtered Buffer/SolventThe Millisolve™ flask can be used as a solvent/buffer reservoir for theHigh Performance Liquid Chromatography (HPLC) or LC system. The Arrayconical bottom of the vacuum flask allows nearly the entire volume offiltered liquid to be utilized.1. Pass required lengths the LC solvent/buffer tubing through the 3-holestopper. If desired, attach a solvent filter/sparger to the ends of thetubing.2. Insert the 3-hole stopper into the flask.3. Connect the LC solvent/buffer tubing to the LC system and heliumsource.4. Pump the buffer through the HPLC or LC system.CleaningClean the system immediately after use. Depending on the solution filtered, you can flush the system with a filtered, volatile solvent (acetone or acetonitrile) or hot water, or disassemble and clean as follows:1. Rinse all components with hot tap water. Clean the porous glass frit of the filter support by back-flushing with tapwater.CAUTION: Do not clean with detergents, as they may introduce contaminants into the system.When cleaning the filter holder with the stainless steel screen filter support, the filter support drops outeasily when the base is inverted. Take care not to mislay or damage the PTFE gasket that lies under thefilter support.2. After rinsing thoroughly with tap water, rinse the components with laboratory-grade water.NOTE: Final rinse water can leave residues that affect critical tests. Use rinse water suitable for your application. 3. Allow components to air dry while disassembled.CAUTION: Do not wipe dry with paper or cloth, as this leaves fibers and lint on the surfaces and also generates electrostatic forces that can attract more dirt. To facilitate drying, flush with a volatile solvent such asacetone or acetonitrile.SpecificationsMaterialsMillisolve™ flask, vacuum cap, filter holder base, funnel ClampFunnel alignment guide3-hole stopper Borosilicate glass Anodized aluminum Silicone PolypropyleneFilter diameter47 mmEffective filter area Approximately 9.6 square centimeters (cm²) (1.5 in²) Funnel capacity300 mLVacuum flask capacity 2 LMaximum pressure948 mbar (28 in. Hg)Approximate dimensionsHeight with vacuum cap Height with funnel Vacuum flask diameter 45.7 cm (18.0 in.)53.3 cm (21.0 in.)12.5 cm (4.9 in.)Ordering InformationThis section lists catalogue numbers for the Millisolve™ Clarification System, replacement parts, and accessories. See the Technical Assistance section for contact information. You can purchase these products online at/products.Product DescriptionMillisolve™ Clarification System XX1614700 Replacement PartsVacuum cap XX1614701Vacuum base XX1514702Funnel, 300 mL XX1014704Clamp XX1014703Vacuum flask with conical bottom, 2 L, ground glass attachment XX1614705Funnel alignment guide ALIGNED05Filter forceps, stainless, smooth-tip XX6200006PPTFE tubing for solvent and buffer, 70 cm (27.6 in.)XT1200000 Accessories for Millisolve™ Clarification SystemBaseXX1514732 Vacuum base with stainless steel screen filter support and gasket,ground glass attachmentFunnelFunnel, 500 mL XX5014704Flasks with ground glass attachmentVacuum filtering flask, 4 L XX1014745Vacuum filtering flask, 1 L XX1514705Vacuum filtering flask with conical bottom, 5 L XX1614706Flask with stopper attachment for use as vacuum trapVacuum filtering flask, 1 L XX1014705Vacuum filtering flask, 1 L, threaded sidearm XX1514706PumpsChemical Duty Pump, 115 V, 60 Hz WP6111560Chemical Duty Pump, 100 V, 50/60 Hz WP6110060Chemical Duty Pump, 220 V, 50 Hz WP6122050FiltersMF-Millipore™ 0.22 µm hydrophilic MCE filter, Triton® surfactant-free, 47 mm, 100/pk GSTF04700Durapore® 0.22 µm hydrophilic PVDF filter, 47 mm, 100/pk GVWP04700Nylon 0.2 µm hydrophilic membrane filter, 47 mm, 100/pk GNWP04700Fluoropore™ 0.2 µm hydrophobic PTFE filter, 47 mm, 100/pk FGLP04700Omnipore™ 0.2 µm hydrophilic PTFE filter, 47 mm, 100/pk JGWP04700MF-Millipore™ 0.45 µm hydrophilic MCE filter, Triton® surfactant-free, 47 mm, 100/pk HATF04700Durapore® 0.45 µm hydrophilic PVDF filter, 47 mm, 100/pk HVLP04700Nylon 0.45 µm hydrophilic membrane filter, 47 mm, 100/pk HNWP04700Fluoropore™ 0.45 µm hydrophobic PTFE filter, 47 mm, 100/pk FHLP04700Fluoropore™ 0.45 µm hydrophobic PTFE filter, unbacked, 47 mm, 100/pk FHUP04700Omnipore™ 0.45 µm hydrophilic PTFE filter, 47 mm, 100/pk JHWP04700LCR 0.45 µm hydrophilic low extractables PTFE filter, 47 mm, 100/pk FHLC04700MiscellaneousMillex®-FA50 filter, 1.0 µm hydrophobic PTFE, 50 mm, 10/pk SLFA05010Tubing, 3/16 in. (4.8 mm) ID x 4.6 ft (140 cm), silicone with Luer adapter XX7100004NoticeThe information in this document is subject to change without notice and should not be construed as a commitment by EMD Millipore Corporation (“Millipore”) or an affiliate. Neither EMD Millipore Corporation nor any of its affiliates assumes responsibility for any errors that may appear in this document.T echnical AssistanceFor more information, contact the office nearest you. In the U.S., call 1-800-645-5476. Outside the U.S., go to our web site at /offices for up-to-date worldwide contact information. You can also visit the tech service page on our web site at /techservice.Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms (“Conditions of Sale”).Millipore, Millex, and Durapore are registered trademarks of Merck KGaA, Darmstadt, Germany.The M initial, Millisolve, MF-Millipore, Omnipore, and Fluoropore are trademarks of Merck KGaA, Darmstadt, Germany.All trademarks of third parties are the property of their respective owners.© 2017 EMD Millipore Corporation. Billerica, MA, U.S.A. All rights reserved.20231184, Rev. 06/17。
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Sample Flow
硬件系统——光学系统原理
激发和收集光信号
激发 组件
激光器：光源，产生 和发射激发光 透镜和棱镜：将激光 器产生的激发光照到 细胞样品上
收集 组件
滤光片：收集相应波 长的从细胞样本上激 发出的光 收集器： 光电倍增管（PMT）
Guava独特的光学系统——双激光共线性
以Guava easyCyte 8HT为例
FL3
软件系统
仪器清洗、维护，数据获取和分析
Muse独特的人机对话方式——触摸屏
MUSE 独特全触屏式控制系统加强操作的简便性 五步触控即完成细胞分析
开机
调整设置
导入样本信 息
获取数据
输出结果!
Overview of Flow Cytometer
Agenda
• Flow Cytometry Introduction • Tips in Flow Cytometry
• FMO
(Fluorescence-minus-one controls)
Step 3：染色流程
临床样本 加入50 µL样本 科研样本 样本+表面抗原抗体
加入20ul mAb 涡旋振荡, 避光孵育15 min 加450ul BDFACS 溶血素
固定 穿膜 胞内抗体
涡旋振荡, 孵育15 min 信号采集和分析
复合染料容易降解 降解因素包括：光照、 甲醛固定剂、温度升高 选择专用固定剂、 APC-H7 批次差异大 不同批次补偿不同
B. Without CD8 APC-Cy7:
Step 2：试剂选择
抗体的选择：
单克隆抗体 直接标记的抗体 特异性克隆 混合性克隆
抗体来源
根据对神经氨酸酶及糖蛋白酶的敏感性不同，CD34 抗体可分为3 类: ① Ⅰ类抗体,这类抗体对上 述两种酶均敏感,需要与CD34 分子的糖类部分及末端唾液酸结合,然而这种结构只存在于部分CD34 的分子中,因此会造成结果不同; ② Ⅱ类抗体,此类抗体只对糖蛋白酶敏感; ③ Ⅲ类抗体,此类抗体对 两种酶均不敏感。Ⅱ类抗体与FITC 结合时会产生静电而影响结合特性,因此目前多采用Ⅲ类抗体 如抗HPCA22 (BDIS) , 581 ( Coulter2Immunotech ) 和Birma2k3 (DAKO)
流式细胞术（Flow Cytometry, 简称FCM）是一种在功能水平上对单细胞或其他生物 粒子进行定量分析和分选的检测手段，它可以高速分析上万个细胞，并能同时从一个 细胞中测得多个参数。
检测样本： 外周血，骨髓，穿刺液，洗脱液，尿液，唾液，实 体组织，培养细胞，微生物，微球、血清、细胞裂 解液等 提供的信息： 相对细胞大小(FSC) 相对细胞颗粒密度和内部复杂度(SSC) 染色过细胞的相对荧光强度(Fluorescence)
Step 1：样品准备
方法一：溶血法 1. 取100 ul抗凝全血； 2. 快速加入2mlNH4CL溶血剂,混匀； 3. 室温孵育10分钟； 4. 4ºC，300g离心10分钟。去上清，加入1mlPBS； 5. 4ºC，300g离心10分钟。去上清，调整细胞浓度至5x106/ml。 溶血素：NH4Cl，草酸 优点：方便，简单，不容易丢失细胞 缺点：细胞形态变化大，荧光背景比较高,影响表面抗原
Step 2：试剂选择
荧光素的选择：
• 根据仪器的配置选择荧光素（488，633，405，375） • 根据抗原表达的强度选择荧光的亮度
• 最小化荧光重叠（FITC、PE）
• 谨慎选择复合荧光染料（PE-cy5、PE-cy7） • 对于荧光背景高的细胞群体的染色，尽量选择红激光激发的染料
Step 2：试剂选择 “Bright” = good resolution sensitivity
洗涤 信号采集和分析
Step 4：仪器质控
Guava Check Beads Daily QC of system performance Levy-Jennings graphs automatically calculated
Step 5：数据获取与分析
• • 空白组：调节PMT电压； 单染组：调节补偿；
• • • • • • •
• ……………
Step 5：数据获取与分析
单染组：调节补偿；
Step 5：数据获取与分析
单染组：调节补偿；
Compensation at 110% Compensation at 15%
Step 5：数据获取与分析
单染组：调节补偿；
Step 5：数据分析
• • • • 画图 设门，设定阴性与阳性群体的界限 确定阳性与阴性细胞群体 统计阳性或阴性细胞群体的百分率，平均荧光值，绝对数 或抗体结合数
Reagent PE Alexa 647 APC PE-Cy7 PE-Cy5 PerCP-Cy5.5 PE-Alexa 610 Alexa 488 FITC PerCP Filter 585/40 660/20 660/20 780/60 695/40 695/40 610/20 530/30 530/30 695/40 Stain Index 356.3 313.1 279.2 278.5 222.1 92.7 80.4 75.4 68.9 64.4
Epigenome
Transcript
Transcriptome
Protein
Proteome
Strength in integrity
Cytology
Cell
What is Flow Cytometry? Flow Cyto Metry
FluidCell NhomakorabeaMeasurement
What is Flow Cytometry?
•
•
阴性对照组：设定界限；
收取足够的细胞数
Tube1: cells+A*-FITC,B*-PE,C*-PerCP
Step 5：数据获取与分析
单染组：调节补偿；
试剂：A-FITC,B-PE,C-PerCP;A*-FITC,B*-PE,C*-PerCP Tube1: cells+A*-FITC,B*-PE,C*-PerCP Tube2: cells+A-FITC,B*-PE,C*-PerCP Tube3: cells+A*-FITC,B-PE,C*-PerCP Tube4: cells+A*-FITC,B*-PE,C-PerCP Tube5: cells+A-FITC,B-PE,C-PerCP Tube6: cells+A-FITC,B-PE,C-PerCP
Step 1：样品准备
方法二：密度离心法提取单个核细胞 常用来分离人外周血单个核细胞（PBMC）的分层液比重是 1.077±0.001 的 聚蔗糖(Ficoll)-泛影葡胺(Urografin)(F／H)分层液。Ficoll是蔗糖的多聚 体，呈中性，犌W水性高，平均分子量为400,000，当密度为1.2g／ml仍未超出 正常生理性渗透压,也不穿过生物膜。红细胞、粒细胞比重大，离心后沉于管 底；淋巴细胞和单核细胞的比重小于或等于分层液比重，离心后漂浮于分层液 的液面上，也可有少部分细胞悬浮在分层液中。吸取分层液液面的细胞，就可 从外周血中分离到单个核细胞。
优点：干净，荧光背景比较低 缺点：容易丢失细胞，操作麻烦
Step 1：样品准备
方法三：贴壁细胞或实体组织处理 Enzymatic digestion : trypsin, collogenase, pronase. Chelating agents - removal of ‘binding’ ions : EDTA, EGTA, TPB. Mechanical : teasing, sieving, aspiration (syringing) and sonication. 要求：单细胞悬液（适当降低浓度）
谨慎选择复合染料
A. With CD8 APC-Cy7 and CD4 PE-Cy7:
Gating scheme CD8 APC-Cy7+ cells CD4 PE-Cy7+ cells
False positives in APC channel reduced in absence of APC-Cy7 False positives in PE channel remain
APC-Cy7
Alexa 700 Pacific Blue AmCyan
7801/60
720/45 440/40 525/50
42.2
39.9 22.5 20.2
Step 2：试剂选择 “Min Spillover” = good resolution sensitivity
Step 2：试剂选择
Step 1：样品准备 Step 2：试剂选择 Step 3：染色流程 Step 4：仪器质控 Step 5：数据采集与分析
The Conversion of Reseach Focus Why more people focus on flow cytometry?
Gene
Genome
DNA Methylation
流式细胞术实用技巧
Sukie Gong Imaging Field Application Scientist– Instrument Platform, Bioscience
26 June, 2015, Wuhan
Agenda
• Flow Cytometry Introduction • Tips in Flow Cytometry
流式细胞仪的基本组成
硬件系统
液流系统 光学系统 电子器件
软件系统
硬件系统——液流系统原理
将样本悬液聚焦在光源的中心处