CD19 CAR T Cell Flow Cytometry Panel

Immunophenotyping of CD19 CAR T Cells

CD19 is expressed on the surface of mature B cells. CD19-targeted chimeric antigen receptor (CAR) T cells have proven clinically effective in treatment of Chronic Lymphocytyic Leukemia (CLL) and B-cell Acute Lymphoblastic Leukemia (B-ALL). Use this validated flow cytometry panel to identify and phenotype CD19-CAR T cells.

 

 

Flow Cytometry Panel for Immunophenotyping of CD19 CAR T Cells

Marker Clone Fluorochrome Catalog #
CD14 134620 Alexa Fluor® 700 FAB3832N
CD45 2D1 PerCP FAB1430C
CD56/NCAM-1 2524C PE FAB24086P
CD8 37006 Alexa Fluor® 405 FAB1509V
CD4 - PE-Cy7™ Available from Novus Biologicals
CD62L/L-Selectin 4G8 Alexa Fluor® 594 FAB9787T
Human CD19 Fc Chimera - Atto 647 ATM9269

*Designate clones independently validated by HLDA.

This multicolor flow cytometry panel was validated on T cells transduced with human CD19 CAR (hCD19 CAR).

 

 

Flow Cytometry Gating Strategy for CD19 CAR T Cell Panel

Pseudocolor flow cytometry plot showing gating strategy for CD19 CAR T cells including CD14, CD45, CD56, CD4, CD8, CD62L and CD19 Fluorokine.

 

Fluorokines can be used with fluorochrome-conjugated antibodies to characterize CD19-CAR-T Cells by multicolor flow cytometry. T cells transduced with a hCD19-CAR were stained with antibodies to CD45 PerCP, CD14 Alexa Fluor® 700, CD56 PE, CD8 Alexa Fluor® 405, CD4 PE-Cy7™, and CD62L Alexa Fluor® 594. To evaluate transduced cells, cells were stained with the Fluorokine Recombinant Human CD19 Fc Chimera Atto 647N Protein (Catalog # ATM9269). Cells were initially gated on singlets and live cells.

 

 

Staining Protocol for CD19 CAR T Cell Panel

Other Supplies Required

  1. 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.
  2. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 10 minutes at room temperature.
  3. Add 5 μL (or previously titrated amount) of each primary conjugated antibody and Human CD19 Fc Chimera. Vortex tubes.
  4. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
  5. Incubate samples for 20 minutes at 37 °C and then 10 minutes at room temperature in the dark.
  6. 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.
  7. Acquire on Flow Cytometer.