Immunophenotyping of CAR T cell effector cytokine production using flow cytometry
Miltenyi Biotec-tested panel 44 (MBTP 44)
Miltenyi Biotec-tested panel 44 (MBTP 44)
This application protocol describes the characterization of CAR T cell functionality in terms of effector cytokine production upon antigen encounter using flow cytometry. To demonstrate the state-of-the-art panel, CAR T cells were co-cultured with antigen-expressing or antigen-negative target cells (GFP+) in presence of Brefeldin A.
Protocol
- Volumes given below are for up to 5×105 nucleated cells. When working with fewer than 5×105 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 1×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- 5×105 CAR T cells were resuspended in 100 µL TexMACS GMP Medium.
- CAR Detection Reagent, human, Biotin was added according to the dilution mentioned in the respective data sheet.
- Cells were resuspended and incubated for 10 minutes in the dark at room temperature (19−25 °C).
- Cells were washed with 1 mL of TexMACS GMP Medium and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 1 mL of TexMACS GMP Medium.
- Appropriate amounts of CAR T cells (e.g. 0.5×105 cells/well) were seeded into a 96-well plate.
- Appropriate amounts of antigen-expressing or antigen-negative cells (e.g. 2.5×105 cells/well) were added to the CAR T cells.
- 1 µg/mL Brefeldin A was added to all samples.
- Co-cultures were incubated for 4 hours.
- Cells were centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- 25 µL of Viobility 405/452 Fixable Dye diluted in PBS was added and incubated for 5 minutes at room temperature (19−25 °C).
- CAR Detection Reagent, human, Biotin was added according to the dilution mentioned in the respective data sheet.
▲Note: If using a PE-conjugated CAR Detection Reagent, directly proceed with step 17. - Cells were incubated for 10 minutes at room temperature (19−25 °C).
- Cells were washed twice with 150−200 µL of PEB buffer and centrifuged at 300×g for 3 minutes.
- Cells were resuspended in 48 µL of PEB buffer containing 1 µL CD3 Antibody, VioBlue, 1 µL CD8 Antibody, PE-Vio 615, and 1 µL Biotin Antibody, PE.
- Cells were incubated for 10 minutes in the dark at room temperature.
- Cells were washed with 150 µL of PEB buffer centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in in 50 µL of PEB buffer and 50 µL of Inside Fix and incubated for 20 minutes in the dark at room temperature (19−25 °C).
- Cells were washed with 100 µL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were washed with 200 µL of Inside Perm and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 47 µL of Inside Perm and 1 µL each of the three intracellular antibodies was added.
- Cells were incubated for 10 minutes in the dark at room temperature (19−25 °C).
- Cells were washed with 100 µL of PEB buffer and centrifuged at 300×g for 5 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 150 µL of PEB buffer before flow cytometric analysis using the MACSQuant 10 Analyzer.
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Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| Viobility™ 405/452 Fixable Dye | - | Viability | VioGreen™ | 525/50 (violet) |
| CD3 | REA613 | T cells | VioBlue® | 450/50 (violet) |
| CD8 | REA734 | Cytotoxic T cells | PE-Vio® 615 | 655-730 (blue) |
| IFN-γ | 45-15 | Effector functions | APC-Vio 770 | 750 LP (red) |
| TNF-α | REA656 | Effector functions | APC | 655-730 (red) |
| IL-2 | REA689 | Effector functions | PE-Vio 770 | 750 LP (blue) |
| CD19 CAR Detection Reagent, Biotin | - | CAR+ cells | - | - |
| Biotin | REA746 | CAR+ cells | PE | 585/40 (blue) |
Additional reagents used
- Phosphate-buffered saline (PBS), pH 7.2
- PEB buffer: PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.
- Deionized or distilled water
- TexMACS™ GMP Medium (# 170-076-306)
- Inside Stain Kit (# 130-090-477)
- Brefeldin A
- Flow cytometer, for example, MACSQuant® Analyzer 10 (# 130-096-343) or MACSQuant X (# 130-105-100)
- (Optional) MACSQuant Calibration Beads (# 130-093-607)
- (Optional) MACS® Comp Bead Kit, anti-REA (# 130-104-693)
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REA734
FC
EUR 356,00
REA689
ICFC
EUR 356,00
REA1297
FC
EUR 1 377,00
REA1298
FC
EUR 1 377,00
