流式细胞仪做血细胞

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流式细胞仪做血细胞

更新时间：2012-09-10

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收集血液（75微升）为含有5毫升的ED TA（10微升0.5股份）和混合立即以防止凝血。保持管冰。

去除红细胞从样品。这可以通过一些手段。在杰克逊实验室，红细胞溶解使用格氏溶液或缓冲氯化铵溶液（确认）。（另外，流式细胞仪销售的产品被称为“流式细胞裂解液”，是用来在染色议定书溶解红细胞和修复细胞。）

细胞应该洗2 - 3表达缓冲区（缓冲辅以1%牛血清白蛋白或5%胎牛血清和含0.05%*）。悬浮颗粒从zui后洗50微升仪缓冲（或如果有一个以上的分析是要做一个样品多达三个独立的染色反应可以设置从单一样本）。

添加50微升10微升的悬浮细胞的抗体溶液，轻轻拌匀。你将需要确定适当的浓度为每个抗体。

孵化为30分钟的冰。

洗细胞2 - 3表达缓冲悬浮在200 - 300微升仪缓冲分析。

生活/死亡歧视，添加10微升碘化（皮）溶液（股票= 10微克/毫升）。如果固定细胞之前的分析，不加皮。

Collect blood （75 microliters） into 1ml PBS containing 5 microM EDTA （10 microliters of 0.5 M stock） and mix immediay to prevent clotting. Keep tubes on ice.
Remove RBCs from samples. This can be accomplished by several means. At The Jackson Laboratory, RBCs are lysed using either Gey's solution or a buffered ammonium chloride （ACK） solution. （Alternatively, Becton-Dickinson sells a product called "FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells.）
Cells should be washed 2-3x with FACS buffer （PBS supplemented with either 1% BSA or 5% FBS and containing 0.05% NaN₃）. Suspend the pellet from the final wash in 50 microliters FACS buffer （or more if more than one analysis is to be done on a single sample - up to three separate staining reactions can be set up from a single sample）.
Add 50 microliters of cell suspension to 10 microliters of antibody solution and mix gently. You will need to determine the proper concentration for each antibody used.
Incubate for 30 minutes on ice.
Wash cells 2-3x with FACS buffer and suspend in 200-300 microliters FACS buffer for analysis.
For live/dead discrimination, add 10 microliters propidium iodide （PI） solution （stock=10 micrograms/ml）. If fixing cells before analysis, do not add PI.