Preparation and precautions for human peripheral blood PBMCs

Preparation process of human peripheral blood PBMC
1. Collect anticoagulated human peripheral blood, dilute it with 1640 serum-free medium or PBS at a ratio of 1:1, mix it upside down or blow it with a pipette gun.
2. In a 15 mL centrifuge tube, add 3 mL of well-mixed Ficoll solution (1.077 g/mL), and then slowly add 2 mL of diluted blood along the wall of the tube, and the separation of the blood and Ficoll solution will be considered as successful.
Note: Remove the Ficoll from the refrigerator at 4℃ half an hour in advance and return it to room temperature. Too high a temperature will result in a poor separation, while too low a temperature will result in too high a density, which will also result in a poor separation. 20-25℃ is the optimal temperature.
3. Carefully transfer the sample to a centrifuge and centrifuge at 500 g for 25 min. 4.
4. Carefully remove the centrifuge tube and aspirate the white membrane layer in the middle, i.e., single nucleated cells. 5.
5. Wash the obtained single nucleated cells with 10 mL of PBS, centrifuge at 250 g for 10 min, and discard the supernatant.
6. Repeat the washing once.
7. Resuspend the cells with cell staining buffer.

Human peripheral blood PBMC flow FSC/SSC charts

Precautions:
1. The temperature at which the Ficoll is used is important; too high or too low a temperature will affect the separation results.
2. Blood samples should preferably be freshly anticoagulated (within 2 h of blood collection), and avoid freezing and refrigeration to maintain cell viability.
3. 50 mL centrifuge tubes are less efficient than 15 mL centrifuge tubes. In order to obtain the largest number of monocytes, it is best to use 15 mL centrifuge tubes and not to use more than one-third of the centrifuge tubes, so that you can aliquot the blood sample and add it to multiple centrifuge tubes.
4. Sorted cell samples can be directly induced to block for cytokine detection, which is the most effective, and the sorted samples can also be frozen and then induced to detect when needed.
5. Samples that are too late to be tested after induction blocking should be frozen and tested within 3 days for best results. It is recommended to use 90% FBS+10% DMSO as the freezing solution. 24 h of freezing and then testing, the results have no significant effect; 3 days of freezing and then testing, CD3 and IFN-γ expression is slightly reduced; 1 week of freezing and then resuscitation testing, CD3 and IFN-γ expression is greatly reduced.

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