Ccr7 fitc

The following Abs were purchased from BD bioscience: human CD3-APC-H7 (Cat#: 560176, dilution: 1:100), CD4-BV421 (Cat#: 562424, dilution: 1:100), CD8-APC (Cat#: 340584, dilution: 1:100), CD45-BV510 (Cat#: 563204, dilution: 1:30), CD45RA-PE (Cat#: 555489, dilution: 1:30), CCR7-FITC (Cat#: 561271, dilution: 1:50) and B7-H3-BV421 (Cat#: 565829, dilution: 1:100); human CCR2-BV421 (Cat#: 357210, dilution: 1:100) Ab was purchased from Biolegend. Expression of human B7-H3 in tumor cell lines and organoid cells was assessed with the 376.96 mAb followed by APC-goat anti-mouse IgG Ab (BD Biosciences, Cat# 550826, RRID: AB_398465, dilution 1:100), and confirmed with another B7-H3 mAb (Clone 7–517; BD Bioscience, Cat#: 565829, RRID: AB_2739369, dilution 1:100)^{32 (link)}. Expression of the B7-H3.CARs was detected using the fusion protein 2Ig-B7-H3-Fc (R&D Cat# 1027-B3) and followed by AF647-goat anti-human IgG (H+L) Ab (Jackson ImmunoResearch Laboratories Inc., Cat# 109-606-088, dilution 1:100); CD19.CAR was detected with the previously described anti-CD19.CAR mAb^{47 (link)}. Samples were acquired with BD FACS Canto II or BD FACS Fortessa using the BD Diva software (BD Biosciences). For each sample, a minimum of 10,000 events were acquired and data were analyzed using Flowjo 10.

At least 5 × 10⁵ events per sample were acquired on a Gallios 10-color flow cytometer (Beckman Coulter). Phenotypic characterization of T-cell subsets was performed using CD3-APC-Cy7, CD39-APC, CTLA-4-PE (all BD Biosciences), CD4-PerCP/Cy5.5, CD8-PE/Cy7, PD-1-APC, PD-L1-PE/Cy7, CCR7-FITC, CD25-AF700, CD127-PerCP/Cy5.5, CD45RA-AF700 (all Biolegend), CD45-PE-eFluor610 (eBioscience) and aqua dead cell stain (Life Technologies).