Human Peripheral Blood PBMC Preparation Process and Precautions

The 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, and mix upside down or by pipetting.

2)  Add 3 mL of well-mixed Ficoll solution (1.077 g/mL) (Ficoll is taken out of the 4°C refrigerator half an hour in advance and returned to room temperature. If the temperature is too high, the separation will not be obvious, and if the temperature is too low, the density will be too large, and the separation effect will also be poor. The separation effect is best at about 20~25°C) to a 15 mL centrifuge tube, then add 2 mL of diluted blood along the tube wall slowly. The obviously stratification of blood and Ficoll liquid represents successful preparation.

3)  Transfer the sample to a centrifuge and centrifuge at 500 g for 25 min.

4)  Take out the centrifuge tube, and suck up mononuclear cells in the middle white film layer.

5)  Wash the obtained mononuclear cells with 10 mL of PBS, centrifuge at 250 g for 10 min then discard the supernatant.

6)  Repeat the wash step once.

7)  Cells are resuspended for later use.

FSC/SSC diagram of human peripheral blood PBMC

Precautions:

1. The temperature of Ficoll is very important. Too high or too low temperature will affect the separation effect.

2. The blood sample should preferably be freshly anticoagulated (within 2 hours after blood collection). To maintain cell viability, freezing and refrigeration should be avoided.

3. In order to obtain the maximum amount of mononuclear cells, it is better to use a 15 mL centrifuge tube rather than 50 mL, and the amount in the centrifuge tube should not exceed one third of the centrifuge tube. Blood samples can be aliquoted and added to multiple centrifuge tubes.

4. The sorted cell samples can be induced to block and directly detected with the best effect. Directly freeze the sorted cell samples. Induce and detect when needed. 

5. For intracellular factors detection, samples that cannot be detected in time after induction are recommended to be stored frozen, and the detection effect is best within 3 days. It is recommended to use 90% FBS + 10% DMSO as a freezing medium. After 24 hours of cryopreservation and retesting, the results were not significantly affected. After 3 days of cryopreservation, the expression of CD3 and IFN-γ decreased slightly. After 1 week of cryopreservation, the expression of CD3 and IFN-γ decreased significantly.

6. Picture of Ficoll after separation: