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流式細胞儀做血細胞
閱讀：1749 發布時間：2012-9-10
收集血液（75微升）為含有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。
去除紅細胞從樣品。這可以通過一些手段。在杰克遜實驗室，紅細胞溶解使用格氏溶液或緩沖氯化銨溶液（確認）。（另外，流式細胞儀銷售的產品被稱為“流式細胞裂解液”，是用來在染色議定書溶解紅細胞和修復細胞。）
細胞應該洗2 - 3表達緩沖區（緩沖輔以1%牛血清白蛋白或5%胎牛血清和含0.05%*）。懸浮顆粒從zui后洗50微升儀緩沖（或如果有一個以上的分析是要做一個樣品多達三個獨立的染色反應可以設置從單一樣本）。
添加50微升10微升的懸浮細胞的抗體溶液，輕輕拌勻。你將需要確定適當的濃度為每個抗體。
孵化為30分鐘的冰。
洗細胞2 - 3表達緩沖懸浮在200 - 300微升儀緩沖分析。
生活/死亡歧視，添加10微升碘化（皮）溶液（股票= 10微克/毫升）。如果固定細胞之前的分析，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.