DIRECT ANTIBODY LABELING

  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 5 minutes at 4ºC. Resuspend cells in Falcon tube in 50µL PBS
  2. Dilute the monoclonals according to results from a previous titration
  3. Add 5µL diluted monoclonal antibody to each tube. Shake tubes gently by hand. Place in an ice bath for 15 minutes in the dark
  4. Add 3mL PBS to each tube. Spin at 200g for 5 minutes at 4ºC
  5. Decant supernatant. Add 400µL 2% paraformaldehyde (pH 7.2-7.4) while vortexing
  6. Cap tubes and store at 4ºC wrapped in foil until ready to run
     

References

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 H.L. Fahey, editors. American Society for Microbiology, Washington D.C., 1986, pp 226-235.