Anti ly6c pacific blue

4T1-GFP cell line was orthotopically injected into the mammary fat pad of 8 week old BALB/c female mice (1 × 10⁵ cells in 50 µl PBS). After 4 weeks, the mice were sacrificed and the tumors were harvested and dissociated in gentle MACS dissociator with enzymatic solution containing 3 mg/ml collagenase (Sigma Aldrich, 11088793001) and 0.1 mg/ml Dnase in RPMI 1640 using the standard program for solid tumors. To receive single cell suspension, the digested cell suspension was filtered through 70 µ strainer with ice-cold PBS. The cells were pelleted and lysed in red blood cell lysis buffer and depleted of CD45+ and EpCAM+ cells as described above. Cells were stained for anti-CD45 FITC (Miltenyi 130-110-658), anti-CD31 FITC (Miltenyi 130-123-675), and anti-EPCAM FITC (Miltenyi 130-117-752), anti-PDPN APC (Biolegend 127410) and anti-Ly6C Pacific Blue (Biolegend 128014). See Supplementary Table 3 for antibodies information. Dead cells were excluded using PI Staining (Sigma Aldrich, P4170). CAF were gated based on PI^-, CD45^-, CD31^-, EpCAM^-, PDPN⁺. For sorting the CAF subpopulation, we used LY6C⁺ as a marker for iCAF and LY6C^- as a marker for myCAF. The cells were sorted using a FACSAria Fusion (BD Biosciences) into FACS tubes containing 1 ml complete DMEM media. The maximum tumor volume of 1000 (mm)³ was not reached in any experiment.

Circulating blood leukocyte subpopulations in mice were assessed using a polychromatic fluorescent bead-based flow cytometry assay (BD TruCount, BD Biosciences, San Jose, CA, USA). Peripheral blood (PB) samples (20 or 50 µl) were stained for 20 min at room temperature with a panel of monoclonal antibodies: anti-CD43-FITC (BioLegend); anti-Ly6G-PE, anti-CD90.2-PE, anti-B220-PE, anti-CD49b-PE (VLA-2 alpha chain), anti-NK1.1-PE (BD Biosciences); anti-MHC-II-(Iab)-PE/Cy7 (BD Biosciences); anti-F4/80-APC, anti-CD11b-AlexaFluor700 (BD Biosciences); anti-Ly6C-PacificBlue (BioLegend). All antibody conjugates were carefully titrated prior to use. Red blood cells were lysed in tubes by adding 450 µl (or 250 µl for 20 µl PB) ammonium chloride-based lysis buffer (BD PharmLyse, BD Biosciences, San Jose) and samples were incubated for another 15 min at room temperature. Data acquisition was performed on a BD LSR II flow cytometer using FACSDiva software (BD). The acquisition flow rate was optimized for different blood sample volumes in the preliminary experiments. At least 30,000 leukocyte events and 3000 TruCount beads were acquired in a single measurement (the threshold was set on SSC and AlexaFluor700 was adjusted for optimal bead visualization).