MBTP 34: Immunophenotyping of CAR T cell persistence and differentiation in blood samples using flow cytometry
Miltenyi Biotec-tested panel 34 (MBTP 34)
Miltenyi Biotec-tested panel 34 (MBTP 34)
This application protocol describes the characterization of CAR T cell persistence and differentiation using flow cytometry. For this, CAR T cells were spiked into whole blood. The data was analyzed using the automated data acquisition and analysis tool Express Mode “CAR_T_Cell_Persistence_h_01”.
Protocol
Surface staining
- Volumes given below were for up to 106 nucleated cells. When working with fewer than 106 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 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
- Cell number was determined.
- Cells were centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- 10 µL of CD19 CAR Detection Reagent was added.
- Cells were mixed well and incubated for 10 minutes in the dark at room temperature.
- Cells were washed adding 1 mL of PEB buffer and centrifuged at 300×g for 5 minutes at room temperature. Supernatant was aspirated completely.
- Step 5 was repeated.
- A master mix with all antibodies was prepared according to the dilutions mentioned in the respective antibody data sheets.
- 100 µl antibody master mix was added to the cells.
- Cells were resuspended and incubated for 10 minutes in the dark at 4 °C.
- Cells were washed by addition of 1 mL of PEB buffer and centrifuged at 300×g for 10 minutes. Supernatant was aspirated completely.
- Cells were resuspended in 100 µL PEB buffer. 1 µL of 7-AAD Staining Solution was added and incubated for 5 minutes in the dark at room temperature right before flow cytometric analysis using the MACSQuant® 10 Analyzer.
Materials
Antibody panel details
| Specificity | Clone | Purpose | Fluorochrome | Detection filter (nm) (laser) |
| 7-AAD Staining Solution | - | Viability | - | 655–730 (blue) |
| CD3 | REA613 | T cells | FITC | 525/50 (blue) |
| CD4 | REA623 | T helper cells | VioGreen™ | 525/50 (violet) |
| CD8 | REA734 | Cytotoxic T cells | APC-Vio® 770 | 750 LP (red) |
| CD45RO | REA611 | T cell differentiation | APC | 655-730 (red) |
| CD62L | 145/15 | T cell differentiation | PE-Vio 770 | 750 LP (blue) |
| CD45RA | REA562 | T cell differentiation | VioBlue® | 450/50 (violet) |
| Biotin | REA746 | CAR T cells | PE | 585/40 (blue) |
| CD19 CAR Detection Reagent | - | CAR T cells | - | - |
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
- 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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