Immunophenotyping of CAR T cell exhaustion using flow cytometry

MBTP 52 = Miltenyi Biotec-tested panel 52

This application protocol describes the analysis of chimeric antigen receptor (CAR) T cell exhaustion by detection of CAR T cell exhaustion markers. To demonstrate the state-of-the-art panel, CAR T cells were generated within 12 days using the CliniMACS Prodigy® T Cell Transduction process. CAR T cell exhaustion was analyzed using Miltenyi Biotec’s automated data acquisition and analysis Express Mode program "CAR T Cell Express Mode Package".

Protocol

Antibody panel for CAR T cell proliferation

SpecificityClonePurposeFluorochromeDetection filter (nm) (laser)
7-AAD*-Viability-655-730 (blue)
CD3*REA613T cellsFITC525/50 (blue)
CD4*REA623Helper T cellsVioGreen™525/50 (violet)
CD8*REA734Cytotoxic T cellsAPC-Vio® 770750 LP (red)
CD366 (TIM-3)REA635T cell exhaustionAPC655-730 (red)
CD223REA351T cell exhaustionVioBlue®450/50 (violet)
CD279 (PD1)PD1.3.1.3T cell exhaustionPE-Vio 770750 LP (blue)
CD19 CAR Detection Reagent, Biotin**-CAR+ cells--
BiotinREA746CAR+ cellsPE585/40 (blue) 

* Alternative CE-IVD/ASR reagents available.
** Alternative CAR Detection Reagents can be used. Choose a CAR Detection Reagent detectable in the PE channel, e.g., CD19 CAR Detection Reagent, human, Biotin (# 130-129-550), CD22 CAR Detection Reagent, human, Biotin (# 130-126-727), BCMA CAR Detection Reagent, human, Biotin (# 130-126-090), CD33 CAR Detection Reagent, human, Biotin (# 130-127-642), CD19 CAR FMC63 Idiotype Antibody, PE, REAfinity™ (# 130-127-342), or CD19 CAR FMC63 Idiotype Antibody, Biotin, REAfinity (# 130-127-345 or # 130-127-349).   

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Materials

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