破膜剂检测MPO和TdT中的问题

niwanmao

标题：破膜剂检测MPO和TdT中的问题
因为小弟是第一次做MPO和TdT，有个M2a患者，第一张是45-ssc图，第二张是骨髓先加cd45 10min，溶血后离心弃上清加BD permeabilizing 1X 500ul 放置10min离心弃上清加MPO的45-ssc图，相差好多，请问如何解决，正确的步骤应该是怎么样？未破膜的45-ssc破膜后CD45-ssc和MPO-FITC
回答者：niwanmao
你的胞内染色步骤有问题，请参见官方的染色步骤：

Add 1 mL of 1X FACS™ Lysing Solution * (Cat. No. 349202) to 100  µ L of activated heparinized whole blood. Incubate 10 minutes at roomtemperature. Centrifuge at 500 x  g  for 5 minutes; decant the supernatant. Add 0.5 mL of 1X FACSPermeabilizing Solution 2. Vortex and incubate for 10 minutes at room temperature. Wash byadding PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN 3 , and centrifuge for 5minutes. Add appropriate volume of fluorescent-conjugated intracellular antibody. Vortex andincubate for 30 minutes at room temperature in the dark. Repeat wash step. Resuspend cells in 1%paraformaldehyde in PBS.
Add 1 mL of 1X FACS™ Lysing Solution to 100 µL of activated heparinized whole blood. Incubate 10 minutes at room temperature. Centrifuge at 500 x g for 5 minutes; decant the supernatant. Add 0.5 mL of 1X FACS Permeabilizing Solution 2. Vortex and incubate for 10 minutes at room temperature. Wash by adding PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN3, and centrifuge for 5 minutes. Add appropriate volume of fluorescent-conjugated intracellular antibody. Vortex and incubate for 30 minutes at room temperature in the dark. Repeat wash step. Resuspend cells in 1% paraformaldehyde in PBS.
按照他们这个步骤，你应该是Solution1和Solution2分别孵育后，再加抗体孵育，而不能用自己的溶血素。

2009年8月12日 0:54

FlowerLin

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niwanmao

FlowerLin 发表于 2013-5-4 13:49

                               
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