Immunofluorescence

Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method

1. Add 100 µl of well-mixed anticoagulated whole blood to the bottom of a labeled tube. (We use EDTA as the anticoagulant.)
2. Add the appropriate primary antibody to each tube. If using unlabeled antibody, a titration is suggested. Conjugated antibody should be used as directed, usually 5 or 20 µl per sample for the pre-diluted test size products from BD Biosciences. Since applications vary, each investigator should titrate reagents to obtain optimal results.
3. Mix well, then incubate in the dark at room temperature for 20-30 minutes.
4. Remove tubes from dark chamber and mix each tube well. Add 2 ml of lysing solution to each tube. Vortex each tube.
5. Incubate at room temperature in the dark for 10-15 minutes.
6. Centrifuge for 5 minutes at 1000 rpm (200 x g).
7. Remove supernatant by aspiration. Then vortex and add 2 ml washing solution to each tube.
8. Centrifuge for 5 minutes at 1000 rpm (200 x g).
9. Remove supernatant by aspiration.
10. 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).
11. Incubate in the dark at room temperature for 20-30 minutes.
12. Remove from the dark.
13. Mix well, then add 2 ml washing buffer to each tube.
14. Centrifuge for 5 minutes at 1000 rpm (200 x g).
15. Remove supernatant by aspiration and vortex.
16. If a third step is not required, proceed to step 18. For third step, add an appropriate third-step reagent (at optimal concentration) to each tube and vortex gently.
17. Repeat steps 11-15.
18. If you will analyze the same day, add 500 µl wash buffer to each tube, vortex, and analyze within 8 hours. If not, fix cells by resuspending in 2% paraformaldehyde buffer* (30 minutes, room temperature), and wash with wash buffer. Resuspend cells in 500 µl wash buffer and store in the refrigerator at 2-8°C for up to 36 hours.
    
    

*Extended storage of fluorescent dyes in paraformaldehyde may affect fluorescence. We recommend using our BD Stabilizing Fixative (cat. no. 338036) for preserving immunofluorescent staining (see the TDS for detailed instructions).

Solutions:

RBC Lysing Solution: BD PharmLyse (cat. no. 555899) or BD FACS Lysing Solution (cat. no. 349202).

Washing Solution: PBS + 0.1% sodium azide + 1% fetal bovine serum.

Paraformaldehyde buffer: 2% paraformaldehyde in PBS.

BD Stabilizing Fixative (cat. no. 338036).