Immunophenotyping of CAR T cell activation using flow cytometry

MBTP 53 = Miltenyi Biotec-tested panel 53

This application protocol describes the analysis of chimeric antigen receptor (CAR) T cell activation. 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 activation was analyzed using Miltenyi Biotec’s automated data acquisition and analysis Express Mode program “CAR T Cell Express Mode Package”.


Antibody panel for CAR T cell activation

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)
CD137REA765T cell activationAPC655-730 (red)
CD154REA238T cell activationVioBlue®450/50 (violet)
CD25REA570T cell activationPE-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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