Immunophenotyping of CAR T cell fitness using flow cytometry

MBTP 60 = Miltenyi Biotec-tested panel 60

This application protocol describes the determination of the cell fitness of chimeric antigen receptor (CAR) T cell products using Annexin V and 7-AAD for discrimination between viable, apoptotic, and dead cells. To demonstrate this state-of-the-art panel for CAR T cell fitness, CAR T cells were generated within 12 days using the CliniMACS Prodigy® T Cell Transduction process.

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Protocol

Staining protocol

Surface staining

The fitness assay should be performed on fresh CAR T cell product.
Prepare 1× Annexin V Binding Buffer freshly on the beginning of each working day.

Cell number was determined.
CAR T cells were diluted using TexMACS GMP Medium to a cell concentration between 2.5×10⁶ cells/mL and 1.8×10⁷ cells/mL.
Two round bottom 96-well plates were labeled for cell cultivation at 37 °C or storage at 4 °C, respectively.
▲Note: The number of viable cells after cultivation at 37 °C or storage at 4 °C is used for the calculation of cell fitness.
▲Note: An additional CAR⁺ reference cell line can be used for the determination of the relative cell fitness.
100 μL prediluted sample was added to each well of the two 96-well plates and filled up to 260 μL with TexMACS GMP Medium. Triplicates are recommended.
One 96-well plate was placed in the fridge at 4 °C and the other 96-well plate in the incubator at 37 °C.
Cells were incubated for 1 hour and 50 minutes.
Cells from both 96-well plates were resuspended gently using a multichannel pipette. 250 μL were transferred to 96-deep well plates.
Cells were centrifuged at 300×g for 5 minutes. 200 μL of the supernatant were aspirated.
Cells were washed by adding 750 μL of 1× Annexin V Binding Buffer and centrifuged at 300×g for 5 minutes. 700 μL of the supernatant were aspirated.
A staining cocktail with all antibodies (and Annexin V and 7-AAD) was prepared in 1× Annexin V Binding Buffer according to the dilutions mentioned in the respective data sheets.
50 µL staining cocktail was added to the cells.
Cells were resuspended and incubated for 10 minutes in the dark at room temperature (19−25 °C).
650 μL of 1× Annexin V Binding Buffer was added to the cells right before flow cytometric analysis using the MACSQuant 10 Analyzer.
Cell fitness was determined using the following formulas:
Normalization of viable cells (per replicate):
Normalized viable cells = (Viable cells [cells/mL] × 10⁶)/debris [cells/mL]
Determination of cell fitness:
Cell fitness = (Mean viable cells 37 °C [cells/mL]/ mean viable cells 4 °C [cells/mL]) × 100
Determination of relative cell fitness:
Relative cell fitness = (Cell fitness sample [%]/cell fitness reference cells [%]) × 100

Tested antibody panel

Antibody panel for CAR T cell fitness

Specificity	Clone	Purpose	Fluorochrome	Detection filter (nm) (laser)
7-AAD*	-	Viability	-	655-730 (blue)
Annexin V	REA1165	Apoptosis	FITC	525/50 (blue)
CD4*	REA623	Helper T cells	VioGreen™	525/50 (violet)
CD8*	REA734	Cytotoxic T cells	APC-Vio® 770	750 LP (red)
CD3	REA613	T cells	APC	655-730 (red)
CD19 CAR FMC63	REA1297	CAR⁺ cells	PE	585/40 (blue)

* Alternative CE-IVD/ASR reagents available.

Gating strategy

Figure 1: Gating strategy showing the analysis of cell fitness of CAR T cells stored at 4 °C. Lymphocytes were identified by a SSC^low gate, which also excluded debris (A). Viable (7-AAD⁻Annexin V⁻), apoptotic (7-AAD⁻Annexin V⁺), and dead (7-AAD⁺Annexin V^+/−) lymphocytes were defined by Annexin V/7-AAD staining (B). V = viable, A = apoptotic, D = dead. Among viable cells, CD3⁺CAR⁺ cells were identified (C) and discriminated in CD4⁺ and CD8⁺ subpopulations (D).

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Figure 2: Gating strategy showing the analysis of cell fitness of CAR T cells cultivated at 37 °C. Lymphocytes were identified by a SSC^low gate, which also excluded debris (A). Viable (7-AAD⁻Annexin V⁻), apoptotic (7-AAD⁻Annexin V⁺), and dead (7-AAD⁺Annexin V^+/−) lymphocytes were defined by Annexin V/7-AAD staining (B). V = viable, A = apoptotic, D = dead. Among viable cells, CD3⁺CAR⁺ cells were identified (C) and discriminated in CD4⁺ and CD8⁺ subpopulations (D).

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Matching products:

130-093-060

Annexin V-FITC

100

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in 1 mL (1)

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7-AAD Staining Solution

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CD8 Antibody, anti-human, APC-Vio
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CD3 Antibody, anti-human, APC, REAfinity™

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CD4 Antibody, anti-human, VioGreen™, REAfinity™

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CD19 CAR FMC63 Idiotype Antibody, PE, REAfinity™

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