Experimental reagent
1. PBS (Dulbecco):
0.10g / L anhydrous CaCl2
0.20g / L KCl
0.20g / L KHPO4
0.10g / L MgCl2.6H2O
8.00g / L NaCl
2.16g / L NaH2PO4.7H2O
Adjust pH to 7.4
2. Growth medium:
RPMI 1640 medium without glutamine
10% FBS
Gentamicin (optional) (10μg / ml)
2mmol / L L-glutamine
1mmol / L sodium pyruvate
3. Cytokinesis (final concentration)
5μg / ml PHA: 1mg / ml mother liquor frozen at -20 ℃
25μg / ml Concanavalin A (conA): 0.4mg / ml mother liquor is stored frozen at -20 ℃
Shanglu mitogens: rehydrate the mother liquor at -20 ℃ and freeze
Experimental procedure
1. Collect 10ml whole blood and add heparin without preservatives (0.2ml heparin / 10ml whole blood). Aseptic operation was maintained throughout the experiment.
2. Add 1ml of 6% glucose to the heparinized blood in a 15ml centrifuge tube, and leave it at room temperature for 20 ~ 30min to sediment red blood cells. The settling time should not be too long, otherwise monocytes will no longer remain in leukocyte-rich plasma.
3. Carefully collect leukocyte-rich plasma on the upper layer of red blood cells from dextran-treated blood.
4. Plasma enriched with leukocytes is centrifuged at 300g for 8 minutes at room temperature to pellet the cells.
5. Discard the supernatant and suspend the cells in 1ml PBS.
6. At room temperature, carefully add the cell suspension to 5ml Ficoll-Hypaque using a sterile Pasteur pipette. This step requires skilled operation to keep the plasma interface clear between the Ficoll-Hypaque layer.
7. Centrifuge at 400g at low temperature (4 ° C) for 30min to pellet the cells. After centrifugation, remember not to damage the layering in the tube.
8. Lymphocytes are in the white turbid band between the plasma and the Ficoll-Hypaque layer, carefully remove and store, discard the red blood cell pellet.
9. Wash the cells once with 5ml PBS, centrifuge at 250g at room temperature for 5min, and suspend the pelleted cells in 3 ~ 5ml growth medium.
10. Transfer all cell suspension to T25 tissue culture flask and add mitogen.
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