Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method Add 100 m l of well-mixed anticoagulated whole blood to the bottom of a labeled tube. Add the appropriate primary antibody to each tube. If using unlabeled antibody, a titration is suggested. Conjugated antibody should be used as directed, usually 20 m l per sample. Mix well, then incubate in the dark at room temperature for 20-30 minutes. Remove tubes from dark chamber and mix each tube well. Add 2 ml of lysing solution to each tube. Individually vortex each tube. Incubate at room temperature in the dark for 10-15 minutes. Centrifuge for 5 minutes at 1000 rpm (200 x g). Remove supernatant by aspiration, vortex, and add 2 mls washing solution to each tube. Centrifuge for 5 minutes at 1000 rpm (200 x g). Remove supernatant by aspiration. If required, add appropriate second step antibody (at optimal concentration) to each tube and vortex gently (if second step antibody is not required, proceed to step 18). Incubate in the dark at room temperature for 20-30 minutes. Remove from the dark. Mix well, then add 2 ml washing buffer to each tube. Centrifuge for 5 minutes at 1000 rpm (200 x g). Remove supernatant by aspiration and vortex. If third step is not required, proceed to step 18. For third step, add Sav-PE to each tube and vortex gently. Repeat steps 11-15. If you will analyze the same day, add 500 m l wash buffer to each tube, vortex, and analyze within 8 hours. If not, add 2% formaldehyde buffer to each tube, vortex, and store in the refrigerator at 2-8º C for up to 36 hours.
Solutions:
Washing Solution: PBS + 0.1% sodium azide + 1% fetal bovine serum.
Formaldehyde buffer: 2% formaldehyde in PBS.
