Cell Surface Staining

STAINING OF CELLS WITH MONOCLONAL ANTIBODIES:
ANALYSIS OF EXTRA-CELLULAR MEMBRANE PROTEINS BY FLOW CYTOMETRY

From Givan, A.L. (2000), chapter in In Living Color: Protocols in Flow Cytometry and Cell Sorting (R. Diamond and S. DeMaggio, eds). Springer, Berlin, pp 142-164.

Before staining cells, you should know about the CRITICAL ASPECTS of staining that are important for success and about the SOLUTIONS you will need.

Add antibody(ies) to tubes/wells

1. Label Eppendorf tubes or template for microtiter plate wells. Add directly conjugated antibody (ies) (for direct staining procedure) or primary unconjugated antibody (for indirect staining procedure) to tubes or wells. Antibodies should have been titered by halving dilutions.The volume of each antibody added is that required to give saturating fluorescence intensity in a final volume of 60ml.

Add buffer to each tube/well (if necessary, to make 20ml volume)

2. Cold S/W buffer is added to bring the total antibody volume to 20 ml. Cover tubes or plates and keep on ice and in dark until cells are ready.

Suspend cells (10^7 to 10^8/ ml)

3. Suspend cells in cold S/W buffer to a final concentration of 10^7 to 10^8 per ml. You need 20 ml of cells for each tube in your staining panel -- plus an extra 50 ml for pipetting margin.

Add blocking solution to cell suspension

4. Mix cell suspension with an equal volume of cold blocking solution.

Add 40 ml of cells/block to each tube or well

5. Add 40 ml of cells with block to each Eppendorf tube or well (already containing the antibody (ies)). Use an automatic pipetter for repeating additions (or use a multichannel pipette for wells if you have enough cells to give you sufficient volume). Be careful not to drag antibody from one well to another.

Swirl plate or vortex tubes

6. Swirl plate or vortex tubes to make sure that antibodies and cells are well mixed.

Incubate cells, for 30 minutes

7. Incubate cells covered, in the dark, at 4°C for 30 minutes. During incubation, use this free time to prepare flow cytometer sample tubes, with 150 ml of cold 2% formaldehyde solution in each. Use test tubes that are appropriate to the sample intake on your flow cytometer. Make one tube for each sample in your experiment.

*Add S/W buffer

8. This is the wash step that is repeated three times. Using an automatic or multichannel pipette, add cold S/W buffer -- 100 ml into each well or 1 ml into each Eppendorf tube.

Resuspend cells

9. Swirl plate or vortex tubes to resuspend cells.

Centrifuge

10. Centrifuge at about 1000 x g for 2 seconds (or just long and fast enough to lightly pellet cells). If using Eppendorf tubes and an angled rotor, place tubes with lid hinges toward the outside.

**Remove supernatant

11. Use a glass pasteur pipette attached to a suction line ( with a trap containing 10% chlorox bleach) to remove supernatant from the cell pellet. If using Eppendorf tubes, the pellet will be on the hinge side of the tube. Be careful to leave the pellet behind (!).

Repeat from * to ** twice more

12. Repeat washing of cells (from step 8 to step 11) two more times (total of three centrifugations).

If direct staining, resuspend cells in 150 ml S/W buffer

13. If using an indirect procedure, skip to step 13x. If using a direct staining procedure, you are almost done now. Add 150 ml of cold S/W buffer to each tube/well. Swirl or vortex to resuspend cells completely.

Add cells to equal volume of formaldehyde solution

14. Use a pipette to remove cell suspension from each tube/well and pipette into one of the prepared test tubes containing 150 ml of cold 2% formaldehyde.

Store for at least 24 hrs

15. Store covered, in the cold and dark, for at least 24 hours to give FSC, SSC, and fluorescence intensities time to stabilize after fixation.

Read on flow cytometer

16. If necessary, roughly dilute each sample with cold S/W buffer from a squeeze bottle so that cells go through the cytometer at 100-1000 cells per second. That’s it!

If indirect staining, add 50ml of secondary antibody to each cell pellet and resuspend cells.

13x. If using an indirect staining procedure, add 50 ml of fluorochrome-conjugated secondary antibody to each cell pellet and swirl or vortex to resuspend. The secondary antibody should be at a concentration that produces saturating fluorescence intensity, but low levels of background.

Incubate for 30 minutes

14x. Incubate covered, in the cold and dark, for 30 minutes.

*Add S/W buffer

15x. Using an automatic or multichannel pipette, add cold S/W buffer -- 100 ml into each well or 1 ml into each Eppendorf tube.

Resuspend cells.

16x. Swirl plate or votex tubes.

Centrifuge

17x. Centrifuge at about 1000 x g for 2 seconds (or just long and fast enough to lightly pellet cells). If using Eppendorf tubes and an angled rotor, place tubes with lid hinges toward the outside.

**Remove supernatant

18x. Use a glass pasteur pipette attached to a suction line ( with a trap containing 10% chlorox bleach) to remove supernatant. If using Eppendorf tubes, the cell pellet will be on the hinge side of the tube.

Repeat from * to ** twice more

19x. Repeat washing of cells two more times (total of three centrifugations for the primary antibody step and now three centrifugations for the secondary antibody step.).

Resuspend cells in 150 ml S/W buffer

20x. You are almost done now. Add 150 ml of cold S/W buffer to each tube/well. Swirl or vortex to resuspend cells.

Add cells to equal volume of formaldehyde solution.

21x. Use a pipette to remove cell suspension from each tube/well and pipette into one of the prepared test tubes containing 150 ml of cold 2% formaldehyde.

Store for at least 24 hrs

22x. Store covered, in the cold and dark, for at least 24 hours to give FSC, SSC, and fluorescence intensities time to stabilize after fixation.

Read on flow cytometer

23x. If necessary, roughly dilute each sample with cold S/W buffer from a squeeze bottle so that cells go through the cytometer at 100-1000 cells per second.