Immunophenotyping of T Cells and B Cells by Flow Cytometry
T cells and B cells are critical mediators of adaptive immunity. This multicolor flow cytometry panel allows for easy identification of T cells and B cells.
Flow Cytometry Panel for Identification of T Cells and B Cells
| Marker | Clone | Fluorochrome | Catalog # |
| CD3 | UCHT1* | Alexa Fluor® 405 | FAB100V |
| CD19 | 4G7-2E3 | PE | FAB4867P |
| CD45 | 2D1 | Alexa Fluor® 647 | FAB1430R |
*Designate clones independently validated by HLDA.
Alexa Fluor® is registered trademark of Molecular Probes, Inc.
This multicolor flow cytometry panel was validated on human peripheral blood mononuclear cells (PBMCs).
Multicolor flow cytometry panel to identify human B Cells.. PBMCs were stained with Anti-Human CD45 Alexa Fluor® 647, CD3 Alexa Fluor 405®, and CD19 PE. B cells indicated by CD19+ cells in histogram plot. All antibodies are validated for flow cytometry.
Other Supplies Required
- PBS
- Flow Cytometry Staining Buffer (Catalog # FC001)
- Fc-block (blocking IgG)
- (Optional) Isotype Control Antibodies
- 5 mL Flow cytometry tubes
- Wash human PBMCs (1 x 106 cells per sample) with 2 mL of Staining Buffer (1X) (Catalog # FC001) or other BSA-containing buffer, by spinning at 300 x g for 5 minutes, using 5 mL flow cytometry tubes. Decant/aspirate supernatant.
- Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 10 minutes at room temperature.
- Add previously titrated amount of each primary conjugated antibody. Vortex tubes.
- (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
- Incubate the mixtures for 30-45 minutes at room temperature in the dark.
- At the end of the incubation, wash with 2 mL of Staining Buffer (1X), by spinning at 300 x g for 5 minutes. Decant/aspirate supernatant.
Recommended Antibody Concentrations
| Marker | Fluorochrome | Recommended Concentration |
| CD3 | Alexa Fluor® 405 | 5 μL/106 cells |
| CD19 | PE | 10 μL/106 cells |
| CD45 | Alexa Fluor® 647 | 0.25-1 μg/106 cells |