- Prepare Hank’s Balanced Salt Solution (HBSS) and heparin solution by mixing a bottle of heparin powder into 50 mL of HBSS. Aliquot 1 mL into each of 50 Eppendorf tubes. (Do this in the hood).
- In mouse house: Warm up mice. Make small nick at the bottom of the tail where the artery is. Bleed no more than 5 drops into the Eppendorf tube with the HBSS and heparin. Stop the bleeding with Kwikstop.
- Back in the lab: Spin down the blood cells at 3000 rpm for 5 minutes. Remove the supernatant.
- Add 1 mL ACK lysis buffer. Let sit at room temperature for 10-15 minutes. Spin down at 3000 rpm for 5 minutes. Remove the supernatant.
- Wash cells by adding 1 mL flow cytometry wash buffer (FCWB). Spin 3000 rpm for 5 minutes.
- Remove supernatant. Transfer cells to 96 well plate, using one well per sample. Add more FCWB to the top of the wells and spin at 1250 rpm for 5 minutes.
- Dump out supernatant. Wash with another 200 uL of FCWB and spin again at 1250 rpm for 5 minutes.
- Add appropriate antibodies using 10 uL of the appropriate dilution. When testing to see if the mice are transgenic Rag or not I usually use:
- Incubate in the refrigerator for 1 hour.
- Wash cells 2X with 150 uL FCWB.
- Transfer to FACS tubes in final volume of 250-300 uL.
Run on the FACScan or FACSCalibur on the 10th floor.