Alexa fluor 700 f4 80

Extracted tumors were minced completely (1–3 mm cubes) by using scalpels in two drops of dissociation solution (1 mg/ml collagenase D (11088858001 Roche, Indianapolis, IN) and 1 mg/ml DNase I (10104159001 Roche) in RPMI 1640) on ice. Tumor pieces were further digested for 45 min with dissociation solution (about 1 g tumor in 10 ml dissociation solution) at 37 °C water bath with manually periodical shaking. The digested tissue suspension was aspirated into a 20 ml syringe with 14 g cannula attached and clumps were triturated 15 times and filtered through 40 um strainer to get the single cell suspension  followed by staining with fluorescent antibodies from Biolegend: PE CD4 (GK1.5, 100407), Brilliant Violet 605 CD8a (53–6.7, 100744), PE CD11b (M1/70, 101207), Alexa Fluor 700 F4/80 (BM8, 123130), Alexa Fluor 488 CD3 (17A2, 100210), APC MHC II (M5/114.15.2, 107613). Stained cells were analyzed using BD LSRFortessa X-20 flow cytometer. Data were collected with BD FACSDiva v 8.0.1 software and were analyzed using FlowJo v 10 software.

MICL knockout mice (Clec12A^−/−) on a C57BL/6 background were produced by Taconic Artemis, USA (see online supplementary figure S1A). Clec12A^−/− and wild-type (wt) mice were bred and maintained at the Medical Research Facility, University of Aberdeen. Mice were separately housed in groups and provided freely with food and water. All experimentation conformed to the terms and conditions of UK Home Office licences for research on animals (PPL 60/4007) and the University of Aberdeen ethical review committee.
Characterisation of MICL expression in 8–12-week-old wt and Clec12A^−/− mice was performed by flow cytometry on cells isolated from the peripheral blood, bone marrow, peritoneal cavity, spleen and lungs, as previously described.14 (link) Antibodies used in these experiments included biotin anti-MICL monoclonal antibody 3097 (link) and isotype control, as well as biotin-Gr-1, FITC-7/4, PE-F480, biotin-F480, PerCpCy5.5-CD11b, PE-CCR3, biotin-NK1.1, PE-CD49, PerCpCy5.5-B220, PE-CD19, PerCpCy5.5-CD3, biotin-CD4, FITC-CD8, biotin-CD11c, PerCpCy5.5-Gr-1, APC-Ly6G, PE-CD11b, PE-Ly6G, PECy7-CD11b, Alexa Fluor 488 anti-STAT5, APC-streptavidin (all from BD Biosciences), and Alexa Fluor 700-F4/80 (BioLegend). Flow cytometry was undertaken using a BD LSRFortessa cell analyser and data analysed using FlowJo.

Spleens were removed from euthanized animals, and single-cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100-μm nylon mesh, followed by dissociation, as we reported previously [39 (link)]. Cells were washed once with complete DMEM media, supplemented with 10% heat-inactivated FBS (hiFBS), then incubated with RBC lysis buffer for 1 min to remove red blood cells, then washed again in complete DMEM media. Cell isolates were prepared, as above, then washed and resuspended in staining medium (1× PBS, 0.5% BSA, 0.02% sodium azide). Ghost Dye Violet 510 was used for live-/dead-cell staining. Leukocytes were labeled with a combination of the following antibodies: PE-Cy5 CD3ε, Brilliant Violet 570 CD4, PE/Cy7 CD8a, APC-Fire750 CD11b, Brilliant Violet 785 CD19, PE/Dazzle 594 DX5, Alexa Fluor 700 F4/80, Brilliant Violet 650 Ly-6C, and Brilliant Violet 711 Ly-6G (BioLegend, San Diego, CA, USA). Gating strategy is as shown in Figure 2a. Data were acquired and analyzed using LSR II flow cytometry and Flowjo software (BD Biosciences, Franklin Lakers, NJ, USA).