Flow Cytometry Laboratory

Intracellular labeling of cells for flow cytometry

Equipment

1. Refrigerated centrifuge which will accept 96-well plate carriers, capable of spinning 2000 RPM. If a centrifuge which will accept plate carriers is unavailable, the procedure can be carried out in 12 x 75 mm polypropylene tubes or microcentrifuge tubes, using aspiration to remove supernates. It is important that the cells and reagents remain chilled at all times, however.
2. Plate vortexer. If not available, the cells can be mixed with wash buffers in the 96-well plate using a multichannel or single channel micropipettor.
3. Flow cytometer.
4. Single and multichannel micropipettors.

Supplies

1. 96-well polystyrene V-bottom plates
2. Micropipettor tips
   
   

Solutions

1. First wash buffer
   

   PBS	450 ml
   Acid-citrate-dextrose	50 ml
   20% NaN3 in PBS	5 ml
   Horse serum (GIBCO BRL)	10 ml

2. Second wash buffer: same as first wash buffer, with horse serum omitted.
3. Primary monoclonal antibodies: Working stocks of 15 μg/ml (in .09% azide) in first wash buffer are kept refrigerated. Working stocks should be maintained free of particulates by filtration through a 0.22μ filter.
4. Second step reagents (2nd) : Anti-mouse immunoglobulin antibodies conjugated to a fluorescent compound such as fluorescein isothiocyanate (FITC), Alexa fluor 488 or 647, phycoerythrin (PE), PE-Cy5.5, and Cy5. Working dilutions depend upon the source of the antibody. They need to be titrated before use. Dilutions vary with the fluorochrome used and the commercial source of the reagent. Working dilutions may vary from 1:100 to 1:4,000. Conjugated antibodies are readily available from several commercial sources. We routinely use the products supplied by Invitrogen and Southern Biotechnology Associates (available through Fisher Scientific in the US).
5. 2% buffered formaldehyde: 25 ml of 37% formaldehyde + 475 ml PBS.
6. 1% Saponin (Sigma S7900-25G) Make up solution in first and second wash buffers.
   

Protocol

1. Prepare cells as usual for flow cytometry. See “Labeling cells with monoclonal antibodies (MAbs) for Flow Cytometry”.
2. Distribute monoclonal antibodies in 96 well V bottom assay plate for 1, 2, or 3 color flow cytometry surface labeling. Add 2 x 106 cells/well and label surface antigens using standard procedures.
3. Wash the cells 2 times in second wash buffer and resuspend in 200 μl 2% formaldehyde in PBS. Incubate 10 minutes at room temperature. Wash once with PBS.
4. Resuspend cells in 200 μl first wash buffer with 0.1% saponin. Add 50 μl of monoclonal antibody to intracellular antigens.
5. Label cells using standard FC protocol, increasing the incubation times to 30 minutes and supplementing wash buffers with 0.1% saponin.
   
   

Note: All antibodies in a single well must be of different isotypes, and be used with isotype-specific second step conjugated antibodies.