英语翻译Flow Cytometry Protocol for Intracellular Staining Using

英语翻译
Flow Cytometry Protocol for Intracellular Staining Using Conjugated Secondary Antibodies
A Solutions and Reagents
1.1X Phosphate Buffered Saline(PBS):Dissolve 8 g NaCl,0.2 g KCl,1.44 gNa2HPO4and 0.24 g KH2PO4in 800 mL distilled water(dH2O).Adjust the pH to7.4 with HCl and the volume to 1 liter.Store at room temperature.
2.Formaldehyde(methanol free)
3.Incubation Buffer:Dissolve 0.5 g bovine serum albumin(BSA)in 100mL 1XPBS.Store at 4°C
B Fixation
1.Collect cells by centrifugation and aspirate supernatant.
2.Resuspend cells briefly in 0.5-1 ml PBS.Add formaldehyde to a final
concentration of 2-4%formaldehyde.
3.Fix for 10 minutes at 37°C.
4.Chill tubes on ice for 1 minute.
C Permeabilization
1.Permeabilize cells by adding ice-cold 100%methanol slowly to pre-chilled cells,while gently vortexing,to a final concentration of 90%methanol.Alternatively,to remove fix prior to permeabilization,pellet cells by centrifugation and resuspend in 90%methanol.
2.Incubate 30 minutes on ice.
3.Proceed with staining or store cells at–20°C in 90%methanol.
D Staining Using Unlabeled Primary and Conjugated Secondary Antibodies
NOTE:Allow for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.Count cells using a hemacytometer or alternative method.
1.Aliquot 0.5-1x106 cells into each assay tube(by volume).
2.Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation.Repeat.
3.Resuspend cells in 100μl Incubation Buffer per assay tube.
4.Block in Incubation Buffer for 10 minutes at room temperature.
5.Add the primary antibody at the appropriate dilution to the assay tubes(see individual antibody data sheet for the appropriate dilution).
6.Incubate for 30-60 minutes at room temperature.
7.Rinse as before in Incubation Buffer by centrifugation.
8.Resuspend cells in fluorochrome-conjugated secondary antibody*,diluted in
Incubation Buffer according to the manufacturer’s recommendations.
9.Incubate for 30 minutes at room temperature.
10.Rinse as before in Incubation Buffer by centrifugation.
11.Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
*Recommended Secondary Antibodies from Invitrogen.
A-11070 Alexa Fluor?488 F(ab')2 fragment of goat anti-rabbit IgG(H+L)(1:1000 dilution)
A-11017 Alexa Fluor?488 F(ab')2 fragment of goat anti-mouse IgG(H+L)(1:1000 dilution)

应用流式细胞仪协议的共轭二抗胞内染色
A解决方案和试剂
1.1倍,磷酸盐缓冲液（PBS）：溶解8克氯化钠,氯化钾0.2克,1.44 gNa2HPO4and0.24克KH2PO4in 800毫升蒸馏水（dH2O）.调整在室温下用HCl和1 liter.Store量pH值to7.4 .
2.Formaldehyde（甲醇免费）
3.Incubation缓冲区：溶于100毫升1XPBS.Store在4 ° C 0.5克牛血清白蛋白（BSA）
乙固定
离心,吸上清1.Collect细胞.
简要0.5-1毫升PBS.Add甲醛2.Resuspend细胞最后
浓度为2-4％的甲醛.
10分钟,在37 ° C的3.Fix
在冰上4.Chill管1分钟.
荤打孔
加入冰冷的100％甲醇慢慢预先冷冻细胞,而轻轻涡旋,以90％的终浓度methanol.Alternatively,消除之前修复通透1.Permeabilize细胞,离心和悬浮颗粒细胞的90％甲醇.
2.Incubate 30分钟,在冰上.
3.Proceed与染色或储存在细胞在90％甲醇20℃.
d染色使用未加标签,小学共轭二抗
注意：允许同型相匹配的单克隆抗体或物种的控制匹配,可使用血细胞计数器或其他方法克隆antibodies.Count细胞抗体.
1.Aliquot 0.5 - 1x106到每个检测管细胞（体积比）.
2.Add 2-3毫升每孵化缓冲区管冲洗的centrifugation.Repeat.
在100μl的孵化缓冲区每检测管3.Resuspend细胞.
在孵化缓冲4.Block 10分钟,在室温下.
5.Add在适当稀释法管的主要抗体（见适当稀释个别抗体数据表）.
6.Incubate为30-60分钟,在室温下.
作为孵化前缓冲7.Rinse通过离心.
在荧光共轭二抗*稀释后,8.Resuspend细胞
孵化缓冲区根据制造商的建议.
9.Incubate 30分钟,在室温下.
作为孵化前缓冲10.Rinse通过离心.
11.Resuspend细胞PBS和0.5毫升的流式细胞仪分析.
*推荐由Invitrogen公司二抗.
阿- 11070 Alexa的福陆公司?488的F（ab'）2片段的羊抗兔IgG（高+长）（1:1000稀释）
阿- 11017 Alexa的福陆公司?488的F（ab'）2片段的山羊抗鼠IgG（高+长）（1:1000稀释）