Mouse Peripheral Blood Single Cell Suspension Preparation Process and Precautions
2022-07-19Author:praise：1
Preparation of mouse peripheral blood single cell suspension
1) Collect peripheral blood samples from C57 mice in anticoagulant tubes.
2) Add 100 μL of fresh blood to the centrifuge tube, then add the flow-through antibody corresponding to 1 Test, mix well, and incubate at 4°C for 30 minutes in the dark.
3) Add 2 mL of 1× red blood cell lysis solution, mix well, and lyse at 4°C for 5 min.
4) Centrifuge at 300 g for 5 min (centrifuge immediately after lysis to prevent damage to cells for too long), discard the supernatant, and obtain a white cell pellet.
5) Wash once with PBS.
6) Add 200 μL of cell staining buffer to resuspend the cells, and use flow cytometry for detection and analysis.
FSS/SSC diagram of peripheral blood of C57 mice
CD45/SSC diagram of peripheral blood of C57 mice
Precautions:
1. There are two types of anticoagulant tubes for blood collection, heparin and EDTA. If red blood cells are directly lysed and then used for subsequent staining experiments, both anticoagulation tubes can be used. If PBMC cells are separated after blood collection, heparin anticoagulation must be used.
2. It is recommended to use 10× ACK Lysis Buffer (E-CK-A105) as the lysate contains no fixative.
3. The 10× ACK Lysis Buffer needs to be diluted with pure water to 1× before the experiment. It is prepared and used immediately. And it is recommended to temporarily store it at 4°C and use it on the same day.
4. For the detection of routine indicators in mouse peripheral blood, samples stored overnight can be used. However, for indicators with relatively low expression levels, it is recommended to use fresh samples for detection.
5. The amount of cells in the peripheral blood of mice is relatively low. It is recommended to staining the cell first and then lysing. This method can reduce the number of washed during sample processing, and reducing the loss of cells and avoiding low content cells go undetectable.