CD19 CAR T Cell Flow Cytometry Panel

Immunophenotyping of CAR T Cells with primary conjugated antibodies and Fluorokines.

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

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

MarkerCloneFluorochromeCatalog #
CD14134620Alexa Fluor® 700FAB3832N
CD452D1PerCPFAB1430C
CD56/NCAM-12524CPEFAB24086P
CD837006Alexa Fluor® 405FAB1509V
CD4-PE-Cy7™Available from Novus Biologicals
CD62L/L-Selectin4G8Alexa Fluor® 594FAB9787T
Human CD19 Fc Chimera-Atto 647ATM9269

*Designate clones independently validated by HLDA.

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

View All Fluorokines™ for CAR T Cell Detection

Flow Cytometry Gating Strategy for CD19 CAR T Cell Panel

Pseudocolor flow cytometry plot showing gating strategy for CD19 CAR T cells.

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.

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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.
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Additional Flow Cytometry Products and Resources

Products:

Isotype Controls
MagCellect™ Cell Selection Kits
Quality Control and Standardization Beads from Novus Biologicals
Fluorokines™ for Chimeric Antigen Receptor Detection

Resources:

Flow Cytometry Handbook
Intracellular Staining with Alcohol Permeabilization Protocol
Intracellular Staining with Detergent Permeabilization Protocol
On-Demand Webinar: Demystifying Multi-parameter Flow Cytometry
On Demand Webinar: Turning Flow Cytometry Upside Down and Inside Out

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