Intracellular labeling of cells for flow cytometry
- 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.
- Plate vortexer. If not available, the cells can be mixed with wash
buffers in the 96-well plate using a multichannel or single channel
- Flow cytometer.
- Single and multichannel micropipettors.
- 96-well polystyrene V-bottom plates
- Micropipettor tips
- First wash buffer
|20% NaN3 in PBS
|Horse serum (GIBCO BRL)
- Second wash buffer: same as first wash buffer, with horse serum omitted.
- 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.
- 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).
- 2% buffered formaldehyde: 25 ml of 37% formaldehyde + 475 ml PBS.
- 1% Saponin (Sigma S7900-25G) Make up solution in first and second wash
- Prepare cells as usual for flow cytometry. See “Labeling cells with
monoclonal antibodies (MAbs) for Flow Cytometry”.
- 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.
- 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
- Resuspend cells in 200 μl first wash buffer with 0.1% saponin. Add 50 μl
of monoclonal antibody to intracellular antigens.
- Label cells using standard FC protocol, increasing the incubation times
to 30 minutes and supplementing wash buffers with 0.1% saponin.
All antibodies in a single well must be of different
isotypes, and be used with isotype-specific second step conjugated