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发表于 2013-11-3 20:19:26
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【从Center for Immunology，Department of Lab Medicine & Pathology，University of Minnesota的主页翻译以下内容，供参考】
体外进行小鼠T细胞多克隆刺激可使用刀豆蛋白A（conA），可同时使CD4和CD8 T细胞活化、增殖、分化。
ConA是一种植物血凝素，可非特异性活化小鼠T细胞，而对B细胞作用微乎其微。本protocol可快速刺激T细胞，一般2天后达到最大的增殖程度。如需产生分化的效应细胞（如CTL效应功能在4－5天时出现），需更长时间的刺激，但此时需减少初始培养的细胞数量，以免细胞增殖过度。
详细步骤：
- Mouse spleen cells are prepared as usual into HBSS. Lymph node cells can also be used. Lyse red blood cells with ACK (this may not be necessary but it typically used) for 5 minutes. Add RP10 media and centrifuge. Resuspend in 5 mls of (warmed) RP10 media, count, and adjust the concentration to 2.5 x 106 cells/ml.
- Thaw Con A stock vials (1 mg/ml) sufficient for the experiment.
- Aliquot 10 ml of cell suspension into a 25 cm2 tissue culture flask. Add freshly thawed Con A to a concentration of 2.5ug/ml or 5 ug/ml (i.e. 25ml or 50ml Con A stock per flask). It is best, if possible, to set up flasks at both concentrations of Con A to correct for variability in stimulation.
- Stand the flask ON ITS END (i.e. not laid flat but with the loosened cap pointing upwards) in the tissue culture incubator. This is to increase cell-cell contact during stimulation.
- Cells proliferate over the next 2 days. Keep an eye on the culture, it should gradually turn yellow as the cells acidify the media, but if it changes color rapidly, this may be contamination. Cell clumps should be very evident from 24 hours onwards. Be careful resuspending the cells before 48 hours as they may be fragile.
- At desired times, remove the activated cells and analyze. For functional assays (e.g. CTL activity), it may be necessary to inactivate residual Con A. This can be done by resuspending the cells in a-Methyl Mannoside (50mM in RP10; stock is 20x). a-MM blocks Con A. Cells can be used immediately after addition of a-MM.
备注
- Cells can also be stimulated in plates (24-well and 96-well plates have been used in experiments). Similar cell densities and doses of Con A can be used.
- Cell densities may need to be reduced if TCR transgenic samples are used (if there are higher percentages of T cells in the sample), to avoid the culture crashing.
试剂
Concanavalin A (also called Con A)
Calbiochem #234567: Mol.Wt. 104,000 (but exists as multimer)
储存液 (100x – 200x) 浓度为1mg/ml，配制方法如下:
- Weigh aliquot of Con A powder into a 50ml centrifuge tube (ConA有毒，避免吸引或泼撒). Aim for around 10mg, but determine exact quantity.
- Add sterile ddH2O to give approximately 1.2 mg/ml (so, for 12 mg Con A, add 10ml water，记得使用组织培养级塑料或玻璃瓶).
- Gently mix and let stand at 4℃for at least 2 hours. Then adjust volume* with ddH2O to give solution of 1mg/ml
- Con A does not go completely dissolve and so the solution will be hazy. Some Con A may therefore be lost in sterile filtration. In short term experiments, we have used Con A stocks which are NOT filtered, but if desired, sterilize through a 0.2mm filter.
- Store at –20℃ in 100ul～1ml aliquots, depending on application.
a-Methyl Mannoside (also called Methyl-a-D-Mannopyranoside, or a-MM)
Calbiochem #462711: Mol.Wt. 194.2
储存液 (20x) 浓度是1M，配制方法如下：
- Dissolve 194.2g a-MM into 800ml RPMI. Once in solution, adjust volume* to 1L.
如果每次用的比较少，可使用下面的量 - Or, for small scale…Dissolve 9.71g a-MM into 40ml RPMI. Once in solution, adjust volume* to 50ml.
- Sterilize through 0.2 mm filter. Store 25 ml aliquots at -20℃.
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发表于 2013-11-3 16:22:38
