Ack red blood cell lysis buffer

After sacrificing the mice, tumors were excised and one-fourth of each tumor sample was taken for tissue digestion for flow cytometry. Each tumor sample was processed as outlined in Irey et al.61 (link) Briefly, tumor samples were minced using surgical scissors, and the resulting tumor slurry was suspended in 10 mL of 1× phosphate-buffered saline (PBS) and incubated on ice. Liberase (Roche) and DNAse I (Alfa Aesar) were added to the tumor slurry and samples were then incubated on a shaker at 37°C and were rotated at 80 rpm for 30 min. Tumor samples were spun down for 5 min at 200 G and the supernatant was aspirated. The samples were then resuspended in 10 mL of DMEM (Dulbecco's Modified Eagle Medium) with 10% fetal bovine serum (FBS) and were passed through a 70-micron cell strainer. The tumor cells were then pelleted, supernatant was aspirated, and 2–5 mL of ACK Red Blood Cell Lysis Buffer (Gibco) was added to each sample. Samples were then vortexed and incubated at room temperature for 5 min. PBS (1×) was added to quench the reaction and samples were spun down at 200 G for 5 min. Tumor cells were suspended in 5 mL of 1× PBS containing 0.5% FBS and 1 mM EDTA and were transferred into 5 mL round-bottom glass tubes. Cell counting and staining were then performed.

BM cells were prepared by centrifuging femur and tibia at 12,000 g for 1 min. All collected cells were treated with ACK red blood cell lysis buffer (Gibco; Thermo Fisher Scientific) before analyses. mAbs against c-Kit (2B8), Sca1 (D7), CD3e (17A2), B220 (RA3-6B2), Ter119 (TER-119), Gr1 (RB6-8C5), CD16/32 (93), CD34 (RAM34), Flt3 (A2F10), CD150 (c15-12f12.2), CD48 (HM48-1), CD45.1 (A20), CD45.2 (104), and Ki67 (B56) were purchased from BD, BioLegend, or eBioscience. Flow cytometric analysis was performed using an LSRFortessa X-20 or FACSCANTO II flow cytometer (BD).
For cell sorting, BM cells were stained with mAbs against CD3, B220, Gr1, and Ter119 lineage markers, followed by negative selection of lineage-negative cells using a magnetic-activated cell-sorting kit (Miltenyi Biotec). Lin⁻-enriched BM cells were stained with mAbs against lineage markers (CD3, B220, Gr1, and Ter119), c-Kit, and Sca1, and KSL cells were sorted using a FACSAria II flow cytometer (BD).
For cell cycle analysis, BM cells were stained using mAbs against c-Kit, Sca1, CD48, CD150, and lineage markers and fixed/permeabilized using Cytofix/Cytoperm (BD). Then, cells were stained with Ki67 and DAPI and analyzed using an LSR Fortessa X-20 or FACSCANTO II flow cytometer. Ki67⁻DAPI⁻ cells were considered to be in G0, Ki67⁺DAPI⁻ cells in G1, and Ki67⁺DAPI⁺ cells in S-G2-M.

T-cells were isolated from total blood according to previously established protocols^{25 (link)}. Whole blood from indicated mice was obtained via submandibular vein puncture or from the orbital sinus and collected into sterile 1.5 mL Eppendorf tubes. 100 μL of whole blood from each mouse was then immediately transferred into a new sterile 1.5 mL Eppendorf tube containing 50 μL 0.5 M EDTA, pH 8. Tubes were kept on ice until all samples were collected for processing. If any signs of coagulation were observed, samples were discarded and re-collected. The entire blood sample for each mouse (150 μL) was transferred into a 15 mL Eppendorf tube containing 5 mL of room temperature (RT) ACK red blood cell lysis buffer (Gibco), briefly vortexed, and incubated at RT for 3 minutes. After incubation the samples were placed back on ice and the reaction was stopped by addition of 8 mL of ice cold T-cell culture medium (RPMI supplemented to final concentrations of 10% FBS, 2 mM L-Glutamine, 0.1 mM 2-mercaptoethanol (BME), and 1% Penicillin-Streptomycin). Samples were centrifuged at 400×g for 4 min at 4 °C, supernatant was discarded by vacuum, and cell pellets were resuspended in 200 μL T-cell culture medium. Samples were kept on ice for a short time until ready for processing by Flow Cytommetry with antibodies as indicated in the figure legend.

Ecotropic virus was prepared by transiently transfecting Phoenix ECO adherent packaging cells with an SFG vector (4.7 μg) and pseudotyped with ecotropic envelope vector pCL-ECO (2.68 μg, PhEco, RRID:Addgene_12371). Transfection was facilitated using GeneJuice (Merck Millipore, 70967), following the manufacturer's instructions.
Splenocytes were extracted as follows: Spleens were pulped and passed through a 70 μm cell strainer. Red blood cells were lysed using ACK red blood cell lysis buffer (Gibco, A1049201) for 5 minutes at room temperature. The reaction was stopped with 15 mL of Hank's Balanced Salt Solution (HBSS, Gibco, J67763.K2). After the cells were pelleted, the splenocytes were passed through a 40-μm cell strainer to obtain a single-cell suspension. Isolated splenocytes were activated with 2 μg Concanavalin A (Sigma, C0412–5MG) and 1 ng murine IL7 (Miltenyi Biotec, 130–094–066) per 1.5 × 10⁶ cells for 24 hours. Cells (5 × 10⁶) were plated on retronectin-coated (Takara, T100B) 6-well plates in 2 mL of retroviral supernatant and spun 800 × g for 90 minutes. The wells were supplemented with 4 mL of complete media R10, incubated for 72 hours at 37°C in 5% CO₂, in the presence of human IL2 (100 iU/mL; GenScript, Z00368).