Lympholyte m gradient

For what concerns Ncr1^KiPtpn11^fl/fl data: splenocytes were obtained by mechanical disruption of the spleen and bone marrow cells were obtained by flushing femurs. Red blood cells were lysed.
For what concerns Ncr1^TgPtpn11^fl/fl data: mice were sacrificed with isoflurane. Cardiac puncture was performed prior to organ removal. Spleens were processed with a GentleMACS Dissociator, filtered through nylon mesh, and layered onto a Lympholyte-M gradient (Cedarlane Laboratories Ltd., Canada). Lymphocytes were harvested from the gradient interface. Livers were perfused before removal, processed in PBS with 1% serum with the GentleMACS, and filtered through nylon mesh. Samples were washed three times and suspended in 40% Percoll and layered on 70% Percoll. Lymphocytes were harvested from the gradient interface and washed once.

Using the IMMUNOPREP™ Reagent System (Beckman Coulter, Brea, CA, USA), 0.1 mL of peripheral blood was hemolyzed and fixed. The number of lymphocytes, T cells, B cells, CD3/B220 double-positive T cells, CD4 T cells, and CD8 T cells was measured using a Cytomics™ FC500 flow cytometer (Beckman Coulter) with Flow-Count™ (Beckman Coulter) as the internal standard. Single-cell suspensions were prepared from the spleen. Red blood cells were lysed, cell suspensions were reconstituted in phosphate-buffered saline, and the number of plasma cells (CD138⁺B220^low) was measured using a flow cytometer [26 (link)]. Kidneys were minced and digested for 30 min at 37°C with 400 U/mL collagenase IV (Worthington Biochemical, Lakewood, NJ, USA). The kidney cell suspensions were subjected to a Lympholyte M gradient (Cedarlane, Burlington, Ontario, Canada) and spun at 2300 rpm for 20 min. The number of lymphocytes, T cells, B cells, CD4 T cells, CD8 T cells, CD3⁺CD4⁻CD8⁻, and double-negative (DN) T cells was measured.

Tumors were isolated from individual mice and processed into single cell suspensions using a mouse tumor dissociation kit with the GentleMACS system (Miltenyi, Auburn, CA) according to manufacturer’s instructions. Cell suspension was passed through a 70 μm nylon strainer to generate a single cell population and TIL separated from tumor cells and debris via a Lympholyte-M gradient (Cedarlane, Burlington, NC). Isolated TILs were incubated with 1:200 dilution of Zombie Aqua (Biolegend, San Diego, CA) to stain for dead cells, washed twice with PBS, incubated with Fc block (Biolegend) for 30 minutes on ice, and then stained for surface antigens indicated by flow panel for 1 hour at 4°C. After washing, cells were fixed with FluoroFix buffer containing 1% paraformaldehyde (Biolegend), washed, and analyzed via flow cytometry. A minimum of 20,000 CD45⁺ events were acquired on the BD FACSCanto II. Flow data were analyzed utilizing FlowJo software (ver. 10.3). Populations were first gated on viable cells using a Zombie Aqua live/dead indicator dye, and CD45⁺ to indicate lymphocyte population. The following sub-gate markers were used for specific populations: CD3⁺CD4⁺ (CD4 T cells), CD3⁺CD8⁺ (CD8 T cells).

B16-F10 tumor material was collected for FACS analysis and single-cell suspension obtained with digestion mixture [0.5 mg/mL collagenase A, (Sigma Aldrich, MO, USA); 0.2 mg/mL hyaluronidase type V, (Sigma Aldrich, MO, USA); 0.02 mg/mL DNase I, (Roche Diagnostic GmbH, Germany); per each 0.25 g of tumor tissue). Red blood cells were lysed using 0.15 M ammonium chloride solution (Sigma Aldrich, MO, USA)]. Dead cells were removed by centrifugation on Lympholyte-M gradients (Cedarlane, Canada). Level of T lymphocytes was determined in homogenous single-cell suspension. To identify the subpopulations of T lymphocytes, the following antibodies were used: PE-Cy™7-CD3e, PE-CD4 and FITC-CD8a (BD, Franklin Lakes, NJ, USA). NK cells were identified with an anti-mouse CD49b antibody (Biosciences, CA, USA). Gate parameters dividing negative from positive cells were chosen based on isotype antibody control probes (Jarosz et al. 2013 (link)).

Mice were sacrificed on the 14^th day of the experiment. Tumors were collected for flow cytometric analysis; single-cell suspension was obtained using a digestion mix (0.5 mg/mL collagenase A, Sigma Aldrich; 0.2 mg/mL hyaluronidase type V, Sigma Aldrich; 0.02 mg/mL DNase I, Roche; per 0.25 g of tumor tissue). Red blood cells were lysed using 0.15 M ammonium chloride (Sigma Aldrich). Dead cells were removed by centrifugation using Lympholyte-M gradients (Cedarlane, Ontario, Canada). To identify the subpopulations of T lymphocytes, the following antibodies were used: PE-Cy7^TM-CD3e, PE-CD4 and APC-CD8a (BD Pharmingen, catalog number: 558431, component: 51-9000790). The titers of antibodies was performed in accordance with the manufacturer’s instructions. Finally, to identify the level of NK cells, an anti-mouse CD49b (pan-NK cells) antibody was used (1 µg/10⁶ cells; eBioscences, catalog number: 17-5971-82). In flow cytometric analyses (BD FACSCanto, BD), gate dividing negative from positive cells was based on isotype antibody control probes: PE-Cy™7 Hamster IgG1_κ, PE and APC Rat IgG2a_κ (BD Pharmingen, catalog number: 558431, component: 51-9000792) or APC Rat IgM (1 µg/10⁶ cells, eBioscences, catalog number: 17-4341-82)^{30 (link)}. The titers of antibodies was performed in accordance with the manufacturer’s instructions.