Macsquant analyzer mq10

CAR T cells were longitudinally monitored in peripheral blood (PB) through multiparameter flow cytometry (MFC) using CD19 CAR detection reagent, as previously described,¹¹ (link) or CD19 CAR FMC63 Idiotype (REA1297) (both from Miltenyi Biotec). CAR T-cell differentiation in infusion product leftovers was assessed by MFC as previously described.¹¹ (link) Antibodies and gating strategies are detailed in supplemental Material. Data were acquired on BD FACSCanto II (BD Biosciences), MACSQuant Analyzer MQ10 or MQ16 (Miltenyi Biotec) and analyzed using FlowJo-v10 and MACSQuantify softwares.

For CAR T-cell differentiation, at least 10⁶ cells from infusion product leftovers were used. Cells were incubated with the CD19 CAR detection reagent (Miltenyi Biotec). After wash, the following antibodies were added: biotin-PE, CD3-FITC, CD4-VioGreen, CD8-APC-Vio770, CD45RO-APC (clone REA611, catalog no. 130–115–556), CD62L-Pe-Vio700 (clone 145/15, catalog no. 130–113–621) and CD197-VioBlue (clone REA546, catalog no. 130–117–353; all from Miltenyi Biotec). Data were acquired on BD FACSCanto II (BD Biosciences) or MACSQuant Analyzer MQ10 (Miltenyi Biotec) and analyzed using FlowJo software (RRID:SCR_008520), version 10. Additional information on differentiation status of CAR T cells within infusion products are reported in Supplementary Fig. S5.