Human Peripheral Blood Single Cell Suspension Preparation Process and Precautions

Human peripheral blood single cell suspension preparation process

1)   Collect human peripheral blood samples in anticoagulant tubes.

2)   Add 100 μL of fresh blood and 2 mL of 1× red blood cell lysate to the centrifuge tube, mix and lyse at 4°C for 10 min.

3)   Centrifuge at 300 g for 5 min (centrifuge immediately after lysis to prevent cells damage), discard the supernatant, obtain white cell pellet.

4)   Wash once with PBS.

5)   Add 100 μL of PBS to resuspend the cells, and directly perform subsequent flow-cytometryantibody staining experiments without counting. Add 1 Test corresponding flow-cytometry antibody to single cell suspension prepared from 100 μL of fresh blood sample and mix well to conduct experiment.

FSC/SSC diagram of human peripheral blood

CD45/SSC diagram of human peripheral blood

Precautions:

1.  There are two types of anticoagulant tubes for blood collection, heparin and EDTA. If red blood cells are directly lysed for cell surface index dyeing experiment, both anticoagulation tubes can be used; if cytokines are detected after blood collection, heparin anticoagulation must be used.

2.  It is recommended to use 10× ACK Lysis Buffer (E-CK-A105) as the lysate without fixative.

3.  The 10× ACK Lysis Buffer needs to be diluted with pure water to 1× and presently dilute it before use. It is recommended to temporarily store it at 4°C and use it on the same day.

4.  For detection of routine indicators in human peripheral blood, samples stored overnight can be generally used. However, for indicators with relatively low expression levels, it is recommended to use fresh samples for detection.

5.  When the supernatant is discarded by centrifugation after the third step of lysis, it is recommended to gently remove the residual liquid with a pipette. If it is poured directly, there will be residual lysate in the centrifuge tube. When 100 μL of PBS is subsequently added to resuspend the cells, the lysate may not be fully diluted, resulting in excessive lysis of red blood cells.

6.  The cell pellet can be resuspended by pipetting gently, or by vortexing at a low speed. 

7.  Human blood samples are recommended to be stained with CD45 for the subsequent data analysis.

8.  When processing human blood samples, it is recommended to set a single-stained tube that stains CD45 only and run the machine at low speed to set the threshold, observe the amount of cells, and circle the lymphocyte population.

9.  Human peripheral blood mainly contains T lymphocytes, and the amount of B lymphocytes is relatively small. If a patient's blood sample is used, the amount of B lymphocytes may be lower.

10. It is not recommended to use overnight samples to detect B lymphocytes, which may lead to low detection signals.