Ack lysing buffer

Cells from spleen and peripheral blood were isolated as described (24 (link)). Spleens were passed through a 70-µm cell strainer (BD Falcon), and red blood cells were lysed using ACK lysing buffer (Gibco Laboratories). Peripheral blood from the retro-orbital sinus was underlaid with Histopaque-1077 (Sigma-Aldrich). The gradient was centrifuged at 2000 rpm without braking for 20 min at 20°C, and mononuclear cells were recovered from the interface. To isolate tumor-infiltrating lymphocytes, tumors were removed, weighed, and chopped into small pieces. Following incubation in 1 mg/ml collagenase IV (Worthington Biochemical Corp.) and 0.01 mg/ml DNase (Sigma-Aldrich) in RPMI containing 10% FBS and 1% penicillin/streptomycin at 37°C for 30 min, digested cells were passed through a 70-µm cell strainer (BD Falcon), and red blood cells were lysed using ACK lysing buffer (Gibco Laboratories). After washing with RPMI containing 2% FBS and 1% penicillin/streptomycin, cells were counted using a hemocytometer.

For cell surface staining, the cells were harvested, washed twice with PBS, and then incubated with an appropriate amount of fluorochrome-conjugated or unconjugated antibodies for 20 min or 60 min at 4°C in 2% PBB (2% BSA in PBS buffer). Fcγ fragment-specific secondary antibodies were used accordingly if necessary. For intracellular staining, the surface-stained cells were fixed and permeabilized in Fixation/Permeabilization solution (BD Biosciences) for 20 min at 4°C followed by two washings with Perm/Wash buffer (BD Biosciences). After intracellular staining was performed, the cells were incubated for 30 min at 4°C in Perm/Wash buffer followed by two more washings with Perm/Wash buffer. To measure the number of CAR T cells that had infiltrated tumor tissues and spleens, the tumor samples were digested with collagenase IV (0.5 mg/ml; Sigma-Aldrich) and deoxyribonuclease (DNase) I (0.2 mg/ml; Sigma-Aldrich) for 30 min at 37°C. Tumor digests and spleens were filtered through 70 μm cell strainers to obtain a single-cell suspension. ACK lysing buffer (Themo Fisher) was used to lyse the red blood cells according to the manufacturer’s instructions. Viability dye was used in each sample. The cells were analyzed by flow cytometry using the LSR II cytometer (BD Biosciences) or BD Accuri C6 flow cytometer (BD Biosciences). The data were analyzed with FlowJo software.

Fifty microliters of peripheral blood was collected once a week from each mouse, after which red blood cells were removed using ACK lysing buffer (Themo Fisher). CAR T cells were assessed with CD3 and CD45 antibodies in blood and tissues from NSG mice and CD3 and CD45 antibodies and FITC-human B7-H3 (4Ig) / B7-H3b protein in tissues from humanized mice or CD3, CD45, CD62L, CD45RA, PD-1, LAG-3, and TIM3 antibodies and quantified using CountBright Absolute Counting Beads (Thermo Fisher) on a BD LSRII flow cytometer.

Fifty μL of peripheral blood was collected once a week from each mouse, after which red blood cells were removed using ACK lysing buffer (Themo Fisher). T cells were assessed with CD3 and CD45 antibodies or CD62L, CD45RA, PD-1, LAG3, and Tim3 antibodies and quantified using CountBright Absolute Counting Beads (Thermo Fisher) on a BD LSRII flow machine.