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发表于 2013-11-4 18:53:23
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dragonhzqsmmu 发表于 2013-11-4 14:54
个人感觉只要是酶消化都会对表面标志有所影响，只不过随着选择的酶的种类、消化时间、温度等条件的影响对待 ...
感谢@dragonhzqsmmu 站友的分享，确实跟酶有些关系。我将该文中的相关章节摘录如下：
To separate individual cells from the plaques, we first used various collagenases that were first tested on PBMCs for preserving the cell surface markers CD3, CD4, CD16, CD45, CD45RA, CD197, CD28, CD27, HLA-DR, and CD38（研究了不同胶原酶对这些标记的影响）.
We treated PBMCs with collagenase XIs（这个酶影响较大） diluted at 5, 10, and 2.5 mg/mL. The lowest enzyme concentration resulted in the reduction of CD8 expression by 90% compared with nontreated cells. Also, the expression of CD4, CD3, CD27, and CD28 was reduced by 40%, 12%, 50%, and 42%, respectively. Because of these strong effects, the ability of this enzyme to liberate cells was not studied further.
Second, we treated PBMCs with collagenase IV at 10, 5, and 2.5 mg/mL and noticed that at the highest concentrations, the expression of CD4 and CD8 were reduced by 29% and 23%, respectively. At 1.25 mg/mL collagenase IV, the surface markers expression was decreased by 4.9±3.4% compared with that on untreated PBMCs. Third, we treated cells with Liberase DL. At the concentrations of 62.5 and 31.5 μg/mL, CD4 was reduced by ≈70%, whereas at 15 μg/mL, the expression of this marker was reduced by only 20±7%. The expression levels of CD3, CD8, CD16, CD45, and HLA-DR were affected marginally.
To liberate cells from atherosclerotic plaques, we applied enzymes at concentrations that did not grossly decrease expression of cell surface molecules in PBMCs. Freshly excised plaques were digested with collagenase IV (2.5 and 1.25 mg/mL) and Liberase DL (25 and 12.5 μg/mL) in the presence of 0.2 mg/mL DNase I for 1 hour at 37°C. The cells were then stained with monoclonal antibodies for CD45, CD3, CD4, and CD8. Collagenase IV at 1.25 mg/mL liberated more lymphocytes than did digestion with 2.5 mg/mL as evaluated by the relative amount of lymphocytes in the gate defined on side scatter (SSC) versus CD45 expression.
On the basis of the relative lymphocyte (CD45＋) yield, we chose treatment with collagenase IV for 1 hour at 37°C（这是最后选择的最佳 条件） as an efficient method to liberate lymphocytes from atherosclerotic plaques while largely preserving cell surface markers. We used this protocol in all our subsequent experiments described below. However, the concentration of the enzyme has to be adjusted for each enzyme batch and in our experiments varied between 1 and 1.25 mg/mL.
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发表于 2013-10-28 15:07:46
