Ack lysing buffer

Mice were sacrificed and liver metastasis infiltrating immune cells were harvested on day 14, which was one week after the dose of GVAX. Each liver was mechanically processed and suspended sequentially first through a 100-μm nylon filter and then through a 40-μm nylon filter, and brought to a volume of 25 mL in T-cell media. The cell suspensions were centrifuged at 1500 rpm for 5 min. The supernatant was aspirated, and the cell pellets were resuspended in 4 mL of ACK lysing buffer (Quality Biological). After 2 min in ACK buffer, the lysing was quenched by adding T-cell media up to a volume of 50 mL. The samples were then centrifuged at 1500 rpm for 5 min, and the supernatant was aspirated. The cell pellet was resuspended in 6 mL of 80% Percoll (GE Healthcare), then overlaid with 6 mL of 40% Percoll, and centrifuged at 3200 rpm for 25 min without braking. The leukocyte layer was removed and resuspended in 50 mL of T-cell media, washed twice and re-suspended in PBS.

To prepare single cell suspensions, NICHE cell reservoir tissue was cut into 2 mm × 2 mm pieces and digested using collagenase/hyaluronidase in DMEM (Stem Cell) at 37 °C for 35 min with orbital shaking at 100 rpm. Digestion was quenched with 2% FBS in PBS and digest filtered through a 40 µm strainer. Cells were pelleted and RBC lysed with ACK lysing buffer (Quality Biological). Cells were washed twice and re-suspended with 2% FBS in PBS. Single-cell suspension was stained with metal-tag viability dye for 5 min and washed with cell staining buffer (Fluidigm), followed by sequential staining of surface and intracellular markers detailed in Supplementary table 4. Next, cells were stained with Cell ID Intercalator Ir (Fluidigm) at 4 °C overnight. The next day, cells were washed and prepared for acquisition with Helios (Fluidigm). The data was analyzed by Cytofbank. Briefly, data was normalized, gated out beads and dead cells, and gated on singlet and CD45⁺ cell population, which was selected to perform a tSNE analysis. The tSNE plots were gated according to the gating strategy described in Supplementary table 5. The ratio of cell populations within CD45⁺ cells was quantified by Cytobank and plotted with Prism (Graphpad).

Bone marrow from 8- to 14-week-old male and female mice was flushed from femurs, tibias, hip bones and humeri. Lin⁻ cells were enriched from bone marrow by RBC lysis (ACK Lysing Buffer, Quality Biological, 118-156-101) followed by negative immunoselection using a mouse Lineage Cell Depletion Kit (Miltenyi, 130-090-858). The Lin⁻ cells were maintained in StemSpan serum-free expansion medium (SFEM) supplemented with mouse stem cell factor (mSCF; 100 ng/ml), mouse interleukin 3 (mIL-3; 10 ng/ml), mouse interleukin 11 (mIL-11; 100 ng/ml), human FLT3 (hFLT3) ligand (100 ng/ml) and penicillin–streptomycin (PenStrep; 1×) for 24 h before electroporation with Cas9-gRNA RNP. Viable cells were enumerated in PBS with 10% Trypan Blue using a Countess II (Invitrogen).

Blood volume collected by cardiac puncture was recorded and used to calculate the normalized number of cells per milliliter. Whole spleens were crushed to obtain a single cell suspension, then red blood cells were removed with ACK lysing buffer (Quality Biological). Femurs and tibias from each mouse were cleaned of connective tissue and spun at 350×g for 5 min to collect marrow before removing red blood cells. Viable cell counts were performed using trypan blue exclusions.

Splenocyte stimulators from 2W-OVA.F1 mice were prepared and their red blood cells were lysed with ACK lysing buffer (Quality biological), followed by 30min incubation with anti-CD90.2 (53–2.1, BD Biosciences) to deplete T cells. Labeled T cells were depleted with two consecutive 35min incubations with rabbit complement (Cedarlane) at 37°C. >40× 10⁶ T-depleted splenocytes were then incubated overnight with 20μg/ml LPS. The following day, 1 × 10⁶ responder cells (Pan-T enriched splenocytes) were plated with 0.5 × 10⁶ stimulators (T-depleted APC’s) in triplicate in a 96-well plate (Corning) and incubated at 37°C overnight. Next, Golgi Plug (BD Biosciences) was added at 1:1000 and incubated for an additional 6h at 37°C. Live/Dead and extracellular staining were performed for 10min and 15min (respectively) on ice, and cells were then fixed with BD Cytofix/Cytoperm according to the manufacturer’s instruction (BD Biosciences). Finally, cells were stained for intracellular IFNg and TNFa and acquired via flow cytometry.

All animal experimental procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital. ELT3-V cells stably transduced with pCMV-luciferase (ELT3-V-luciferase) were transfected with Scr siRNA and Etv2 siRNA. 24-h post-transfection, 1 × 10⁶ cells were injected into female immunodeficient C.B17 SCID mice (Taconic) via lateral tail vein injections. Before imaging, mice were injected with the RediJect D-luciferin bioluminescent substrate (Cat. no. 770504; Perkin-Elmer). Bioluminescent signals were recorded at 4, 24, and 48 h using an In-Vivo Xtreme imaging system (Bruker). Net luminescence intensity in the chest area was assessed using Bruker molecular imaging (MI) software (v7). Murine blood was collected at the end of the experiment by aortic puncture, and red blood cells were lysed using an ammonium–chloride–potassium (ACK) lysing buffer (Quality Biological). In addition, murine lung tissues were harvested, minced, and enzymatically digested (300 units/ml Collagenase 4; Worthington Dorchester). DNA was extracted from blood and mouse lung tissues using a Blood and Tissue DNA kit (QIAGEN). Rat and mouse DNA were quantified by RT-qPCR using rat- and mouse-specific primers included in Table S1 (Walker et al, 2004 (link); Yu et al, 2009 (link)).