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流式細胞儀做血細胞
閱讀：1762 發(fā)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(chǎn)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(fù)細胞。）
細胞應(yīng)該洗2 - 3表達緩沖區(qū)（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(yīng)可以設(shè)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(dāng)?shù)?/span>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.