Ccr7 clone g043h7

A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10.7 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g. nontransduced T cells or B7-H3-negative cell lines) served as gating controls. CAR detection was performed using F(ab’)₂ fragment-specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), and CD45RO (clone UCHL1, BD Biosciences). LM7 and the negative control leukemia cell line BV173 were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). Cells were additionally stained with DAPI (BD Biosciences) to gate for live cells.

A single-cell suspension of PBMCs was prepared to determine the cellular phenotype by flow cytometry. The following monoclonal antibodies (mAb) were used: CD3 (clone HIT3a), CD4 (clone A161A1), CD25 (clone BC96), TNF (clone MAb11) TNFR1 (clone W15099A), TNFR2 (clone 3G7A02), CD45RA (clone HI100), and CCR7 (clone G043H7), All the mAbs were provided by BioLegend (San Diego, CA, USA). PBMCs were stained for 30 min at 4 °C in the dark.
To determine the intracellular expression of FOXP3, PBMCs were washed, fixed, and permeabilised with the Kit True Nuclear™ Transcription Factor Buffer Set (BioLegend). Then cells were washed twice and stained with mAb to identify FOXP3 (clone 206D). Finally, cells were washed and resuspended in 1% paraformaldehyde.
Cells used for Fluorescence Minus One (FMO) condition were stained and acquired in parallel to identify staining background levels, and dead cells were omitted using Zombie Red™ (BioLegend) viability kit.
The data were collected using a FACS Aria II (BD Biosciences, San Jose, CA, USA) and then analysed by FlowJo v10.2 (FlowJo LLC, Inc, Ashland, OR, USA). In each case, 50,000 events of the live cell's gate were acquired per sample.