Phenotypic analysis of cells using immunofluorescence is the most widely used application of flow cytometry. Antigen expression, both extracellular and intracellular, can be quantitated using fluorescently-labelled antibodies or ligands directed towards these biomolecules. Over the past few years, the introduction of novel fluorochromes with unique spectral (excitation and emission) properties has facilitated the use of multicolour immunofluorescence analysis permitting the identification and characterisation of specific subpopulations of cells. Furthermore, this has also facilitated the combination of flow cytometric assays for both phenotype and function.

There are many protocols for staing cells with monoclonal antibodies for immunofluorescence analysis by flow cytometry. The protocols outlined below are examples and can or may need to be modified for optimal staining of different cell types. Cells can be stained in FACS tubes or in 96-well plates. Tubes allow for larger volumes of wash buffer to be used, which may be important when lysing red blood cells, but the 96-well plate allows for faster, easier handling of larger numbers of samples.

Directly-conjugated antibodies require only a single-step incubation and are therefore easier and faster to use. They are also advantageous when performing multicolour immunofluorescence analysis or using several antibodies of the same isotype. Indirect immunofluorescence staining uses unconjugated primary antibodies, which recognise the epitope/antigen of interest and then a second-step staining with a fluorescently-conjugated secondary antibody that will bind to the primary antibody. Care must be taken when using indirect staining with multiple antibodies to avoid cross-reactivity between secondary antibodies and different primary antibodies. It may be beneficial to combine direct and indirect staining techniques with conjugated and unconjugated primariy antibodies.

Considerations should also be given to the choice of fluorochrome. Try to match the antibody-fluorochrome combination to the antigen density so use brightest fluorochromes to detect those antigens expressed at the lowest levels.

A. General Immunofluorescence Staining Protocol using Directly Conjugated Antibodies:

1. Prepare single cell suspension and wash in staining buffer (PBS, 2% FCS, 0.1% azide).
2. Centrifuge (300 xg, 5 min, 4oC.), discard supernatant and resuspend to 1 x 107 cells/ml with staining buffer.
3. Aliquot 100 ul of cells into a 12 x 75 mm polypropylene FACS tube.
4. Add 5ul/tube of blocking antibody (e.g. Fc Block).
5. Vortex and incubate for 2 min at room temp.
6. Add monoclonal antibodies, vortex and incubate for 30 min at 4oC (on ice) in the dark.
7. Add 2 ml of staining buffer, vortex and centrifuge (300 xg, 5 min, 4oC).
8. Discard supernatant, resuspend cells in 100 ul staining buffer and add 20ul second antibody.
9. Vortex and incubate for 30 min at 4oC on ice in the dark.
10. Add 2 ml staining buffer, vortex and centrifuge as previous.
11. Discard supernatant.
12. Wash again in 1 ml staining buffer and resuspend in 500 ul staining buffer for FACS analysis.
13. Keep cells on ice prior to analysis.
14. Cells may be centrifuged and fixed in 1 ml of 1% paraformaldehyde (in PBS) at 4oC for analysis next day.

NOTES:
Use buffers without Phenol Red
The blocking antibody step (4 and 5) is optional but should be included if cells express high levels of Fc receptors which will contribute to non-specific binding and background fluorescence.

1. Prepare single cell suspension and wash in staining buffer (PBS, 2% FCS, 0.1% azide).
2. Centrifuge (300 xg, 5 min, 4oC.), discard supernatant and resuspend to 1 x 107 cells/ml with staining buffer.
3. Aliquot 100 ul of cells into a 12 x 75 mm polypropylene FACS tube.
4. Add 5ul/tube of blocking antibody (e.g. Fc Block).
5. Vortex and incubate for 2 min at room temp.
6. Add primary antibody, vortex and incubate for 30 min at 4oC (on ice) in the dark.
7. Add 2 ml of staining buffer, vortex and centrifuge (300 xg, 5 min, 4oC).
8. Discard supernatant, resuspend cells in 100 ul staining buffer and add 20ul second antibody.
9. Vortex and incubate for 30 min at 4oC on ice in the dark.
10. Add 2 ml staining buffer, vortex and centrifuge as previous.
11. Discard supernatant.
12. Wash again in 1 ml staining buffer and resuspend in 500 ul staining buffer for FACS analysis.
13. Add secondary antibody, vortex and incubate for 30 min at 4oC (on ice) in the dark.
14. Wash twice as previous and resuspend in 500 ul FACS buffer.
15. Keep cells on ice prior to analysis.
16. Cells may be centrifuged and fixed in 1 ml of 1% paraformaldehyde (in PBS) at 4oC for analysis next day.

1. Single-Colour Immunofluorescence:

2. Two-Colour Immunofluorescence:

3. Three-Colour Immunofluorescence:

4. Four-Colour Immunofluorescence:

5. Five-Colour Immunofluorescence:

6. Six-Colour Immunofluorescence: