人外周血单核细胞的分离、培养
淋巴细胞是这样分离的：
1、无菌采集静脉血2ml注入盛有2ml hank’s液(含100u/ml肝素)的试管中，混匀。
2、吸取淋巴细胞分层液2ml加入离心管中，再将稀释抗凝血液小心沿管壁加至分层液上，应注意保持两者界面清晰。(关键)
3、室温2000r/min离心20min。管内可分为四层。
4、将上层淡黄色的稀释血浆取出于洁净试管中-20℃保存。
5、用毛细吸管轻轻吸出乳白色的单个核细胞，加入另一支已含有2~5mlhank’s液的离心管中，混匀。
6、室温下,1500rpm离心10min。
7、弃上清，重复洗涤一次。用*rpmi-1640 1ml定容，计数细胞，调整细胞悬液至所需浓度：一般每ml血可分离出1~2×106个单个核细胞。
首先搞清楚“外周血单个核细胞 peripheral blood mononuclear cells 即pbmc”和“单核细胞 monocyte”是两个不同的概念，前者包括单核细胞、t细胞、b细胞等。体外培养能贴壁的，应该是单核细胞，不贴壁的当然是t、b等其他细胞了。你也可以用密度梯度离心法分离pbmc后，再用抗cd14的免疫磁珠分离单核细胞，优点是分离出的单核细胞较均一，活性好。加gm-csf、il-4、lps等培养后可分化为树突状细胞dc。估计你是想获得dc吧。
以下是我用过的分离、保存pbmc的protocol，供参考。可根据所需体积作相应调整。
b. separation of peripheral blood mononuclear cells (pbmc)
1.fill in all required information on data sheet “cryopreservation of peripheral blood mononuclear cells.”
2.remove the rubber stoppers from the vacutainer tubes. wipe off the top of each tube with an alcohol swab.
3.transfer the blood to an appropriate size flask using a sterile pipet. dilute the blood 1:1 with rpmi 1640. use approximay 10 ml rpmi 1640 to rinse the vacutainer tubes, and combine with the rest of the blood.
4.transfer 13 ml ficoll (warmed to room temperature) to each 50 ml centrifuge tube needed to process the entire sample.
5.carefully layer the diluted blood over the ficoll.
6.centrifuge at 2000 rpm for 15 minutes with centrifuge brake off.
7.collect the cell band at the interface from each centrifuge tube, and transfer it to fresh centrifuge tube(s). add rpmi 1640 for a final volume of 45 ml in each centrifuge tube. centrifuge at 2000 rpm for 5 minutes. remove the supernatant, combine the cells in all of the tubes into one tube in 45 ml rpmi 1640. count the cells.
c. cryo-preservation of isolated pbmc
1.centrifuge cells at 2000 rpm for 5 minutes. remove the supernatant.
2.resuspend the cells at 20 x 106 cells/ml in freezing solution (50% fcs, 40% rpmi 1640, and 10% dmso).
3.transfer 1 ml of cells into each 1.8 ml cryovial that is labeled with donor’s name, hospital number, date, and cell type.
4.transfer all cryovials to a pre-cooled freezing chamber and freeze using the appropriate program.
5.once the program is complete, transfer the cryovials to a liquid nitrogen freezer