Human or Mouse Regulatory T Cell Staining Kits From eBioscience

The regulatory T cell staining kits from eBioscience are designed to identify the subpopulation of T cells called T regulatory cells. These cells are characterized by expression of CD4 and high levels of CD25 on the surface and, specific for T regulatory cells, intracellular presence of the transcription factor FOXP3.

The kits contain reagents for 25 or 100 analyses if 1 million cells and 20 µl of antibody are used per test. In my experience, it was sufficient to use 500,000 cells and 8 µl of each of antibody when cells were obtained from blood after PBMC isolation and 1 day after stimulation. All the steps of staining of cells were performed on ice, with cold buffers and with the centrifuge set to +4ºC. Samples should be prepared such that each has its own isotype control.

After the standard procedure of surface staining with anti-CD4 and anti-CD25 antibodies, the manufacturer gives an option to wash the cells in PBS. However, I found that the intracellular staining was better when cells were washed in their staining buffer. Additionally, for murine cells, it was necessary to incubate them with Fc blocking reagent for 15 minutes in order to lower non-specific staining. For human cells, it is optional to use rat serum to lower non-specific binding; I did not find this necessary in my experiments. After the washing step, for human PBMCs, or after Fc-block incubation, for murine cells, cells were fixed and permeabilized in the fix-perm solution (supplied). This solution must be freshly prepared by diluting it 4x in the supplied diluent. Cells then have to be incubated on ice in 1 ml of this buffer for 30 to 60 min; I did this for 45 min. After that step, cells need to be washed twice in 2 ml of permeabilization buffer which is prepared from concentrate by 10x dilution in water. After the last wash, cells were resuspended in 80 µl of staining buffer and anti-foxp3 or an isotype control was added. Cells were incubated for another 45 minutes on ice. After the staining, cells were washed twice with 2 ml of permeabilization buffer and after the last wash, resuspended in 0,5 ml of staining buffer.

Analysis is performed on a flow cytometer. The cells stained with the isotype control antibodies, to which the laser voltage and the lymphocyte gate are set, should be read first. Following the setting of these parameters, the foxp3 stained cells can be read.

The conditions provided above are good for PBMC obtained from blood or for these cells following short stimulation in in vitro conditions. It is possible that the amount of antibody added should be changed if the time and the magnitude of cell responses are higher. For these cases, antibody titrations should be performed.