Cytofix cytoperm fixation and permeabilization solution

PBMC were stained with antibodies as stated in Supplementary Table 2A for 20 min in the dark at 4 °C in PBS (5% FBS), 2 mM EDTA (FACS Buffer). For CD85j and CD244, cells were single stained with CD85j, followed by CD244 before adding the master mix to the cells (each staining is 20 min in the dark at 4 °C) (Fig. S1).
CD107a was added at the start of the stimulation. After stimulation, cells were stained with surface markers before being fixed and permeabilized for 20 min in 4 °C with BD CytoFix/CytoPerm Fixation and Permeabilization Solution (BD Biosciences). The cells were washed twice with 1× Perm/Wash Buffer (BD Biosciences). PBMCs were stained in 1× Perm/Wash with antibodies as stated in Table S2A for 30 min in 4 °C and washed twice before re-suspending in 100 μL FACS buffer.
Samples were acquired using BD LSRII/Fortessa/FACSSymphony flow cytometer using automatic compensations.

To measure intracellular T cell cytokine and granzyme B production, CAR T cells were co-cultured at a 1:5 with U937-CD33^high tumor cells. Approximately 16 hours later, a 500X Protein Transport Inhibitor cocktail was added to the cell culture (eBiosciences), and 6 hours later, cells were collected, stained with Fixable Yellow Dead stain (ThermoFisher Scientific), and fixed and permeabilized with BD Cytofix/Cytoperm Fixation and Permeabilization Solution (BD Biosciences) as per the manufacturer’s suggested protocol. Cells were counted and stained with the following human antibodies: cetuximab-conjugated in-house with PE, anti-CD4 (RPA-T4), anti-CD8 (SK1), anti-IL-2 (MQ1-17H12), anti-TNF-alpha (MAb11), anti-IFN-gamma (4S.B3), and anti-granzyme B (QA16A02).

Details of antibodies and viability dyes are shown in Online Supplementary Table S1. All antibodies were used at pre-determined optimal concentrations. Hematopoietic stem and progenitor cells were analyzed as previously described.^{28 (link)} Cell acquisition and analysis were performed on a BD LSRFortessa (BD Biosciences, San Jose, CA, USA) using BD FACSDiva™ software (BD Biosciences). Cell sorting was performed on a BD FACSAriaII cell sorter (BD Biosciences). Cells used in cell sorting experiments were c-Kit-enriched (MACS Miltenyi Biotec, Bergisch Gladbach, Germany) and were stained with specific antibodies following initial Fc-block incubation. Gates were set using a combination of fluorescence minus one controls and populations known to be negative for the antigen.
For HSC cell cycle staining, BM cells were c-Kit-enriched and stained following initial Fc-block incubation. Stained cells were then fixed and permeabilized using BD cytofix/cytoperm fixation and permeabilization solution (BD Biosciences). Cells were stained with Ki-67 PE (BD Biosciences) overnight. Cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) (0.5 mg/mL) (ThermoFisher Scientific, Waltham, MA, USA) for one hour before analysis.

Cell suspensions were resuspended in PBE and incubated on ice for 30 min with fluorescently labeled antibodies (Table S2) along with 1 mg/ml anti-CD16/32 (24G2; eBioscience).
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.