1. Collect cells from culture or tissue. Make a single cell suspension of ~1x106 cells. Wash cells by adding 3mL PBS, vortex gently and centrifuge at 200g for five minutes at 4oC. Resuspend cells in Falcon tube in 50 uL PBS.
2. Dilute the monoclonals according to results from a previous titration.
3. Add 5uL diluted monoclonal antibody to each tube. Shake tubes gently by hand. Place in an ice bath for 15 min. in the dark.
4. Add 3mL PBS to each tube. Spin at 200g for five minutes at 4oC.
5. Decant supernatant. Add 400uL 2% paraformaldehyde (pH 7.2-7.4) while vortexing.
6. Cap tubes and store at 4oC wrapped in foil until ready to run.
Jackson, A.L, Warner Preparation, Staining and Analysis by Flow Cytometry of Peripheral Blood Leukocytes. In Manual of Clinical Laboratory Immunology. N.R. Rose, H. Friedman and J.L. Fahey, editors. American Society for Microbiology, Washington, D.C., 1986, pp 226-235.