本帖最后由 钢蛋儿 于 2014-6-15 14:59 编辑
我天天提取PBMC，怎么可能不知道……………我的意思的，这个PDF没有说清楚，到底是冻存的全血还是冻存的PBMC…………………
因为Isolation这部分一开始就针对全血进行台盼蓝染色、计数，应该先提取出PBMC再染色计数的…………………
Isolation
1. Collect blood via venipuncture directly into CPT tube(s).
2. Store CPT tube(s) at room temperature if they cannot be processed immediately; cell degradation will
occur if tubes are stored for more than four hours.
3. Centrifuge CPT tube(s) at 1800 x g (approximately 2800 rpm on a Sorvall RT6000 centrifuge) for 20
minutes at room temperature. Be sure that the tubes are not loaded in the outer positions of the
carriers, as this may cause the rubber tops of the tubes to contact the rotor and come off.
4. After centrifugation, bring the CPT tube(s) to a biological safety cabinet and carefully open the tops.
Using a 5 ml pipette, gently pipette the plasma up and down against the gel plug to dislodge cells that
are stuck to the top of the gel. Avoid vigorous pipetting that would disintegrate the gel plug itself.
5. Transfer the cell suspension from the CPT tube(s) to a 50 ml conical polypropylene tube, pooling the
cells from each tube if there are multiple CPT tubes per donor. Add cRPMI to a total of 40 ml.
6. Remove a 10 ml aliquot of cell suspension for counting as per Cell Counting With a Hemacytometer,
and Determining Cell Viability With Trypan Bluesection, following.
7. Centrifuge 50 ml tubes at 250 x g (approximately 1200 rpm on a Sorvall RT6000 centrifuge) for seven
minutes at room temperature.
8. Count cells with hemacytometer while tubes are centrifuging.
9. When centrifugation is complete, aspirate the supernatant and gently flick the tube with a finger to
break up the pellet.
10. a) If cells are to be used fresh: Resuspend the cells at 5 x 10
6
viable lymphocytes per ml in cRPMI.
Proceed with functional assay as per protocol..
b) If PBMCs are to be cryopreserved: Prepare reagents for cryopreservation in advance. If this is not
possible, make sure the cell pellet is disrupted and store the cells on ice until all reagents are
prepared. Resuspend the cells as per Cryopreservation of PBMCssection, following.
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发表于 2014-6-11 12:08:10
发表于 2014-6-11 22:26:04
只是我也特别关心PBMC冻存这个话题所以才精读啦