Gr1 bv510

Tumor-infiltrating lymphocyte isolation was performed as previously described.9 (link) Cells were blocked with antimouse CD16/32 for 30 min at 4°C and then stained with CD45 APC (Cat #187357), CD4 APC-Fire 750 (Cat #244105), CD8 PercpCy5.5 (Cat #277115), Foxp3 Alexa488 (Cat #227489), Granzyme B Pacific-blue (Cat #267707), Gr1 BV510 (Cat #238839), CD11b Alexa700 (Cat #259438), CD38 PE-Cy7 (Cat #216741), and CD206 PE (Cat #225355) from BioLegend. Samples were run on a Gallios (BD Biosciences) flow cytometer and analyzed with Kaluza Analysis Software.

Tumors were harvested, weighed, and processed to obtain single-cell suspensions. In brief, tumor tissues were dissociated and digested with 250 µg/mL of Liberase (Roche) and incubated for 30 min at 37 °C, while shaking at 105 rpm for proper digestion. Fetal bovine serum was then added to stop the reaction, and samples were filtered and washed with PBS + 2% FBS. The cell count per sample was performed, then samples were stained using fluorochrome-conjugated antibodies from BioLegend including: CD45 Pacific blue, cat# 103126; CD4 BV605, cat# 100451; CD8 PE, cat# 100707; CD49b APC, cat# 108910; Foxp3 Alexa488, cat# 126406; TIGIT PE-Cy7, cat# 142108; Gr1 BV510, cat# 108437; CD11b APC-fire750, cat# 101262; F4/80 Alexa700, cat# 123130; CD206 PercpCy5.5, cat# 141716. In alternate panels, a set of other antibodies was also used including: CD4 FITC, cat# 100406; CD8 PErcpCy5.5, cat# 100734; Gr1 APC, cat# 108412; CD11c BV510, cat# 117337; and PVR (CD155) PE, cat# 132206. Samples were run on the Attune flow cytometer, and data were analyzed using Flow-Jo 10 software.

Blood was collected in heparinized tubes and before centrifugation, 10 μl of blood sample were taken for determining the absolute number of leukocytes using CD45-FITC antibody (BioLegend, Fell, Germany) and AccuCount particles (Spherotech Inc., Lake Forest, IL, USA). After centrifugation plasma was taken and the cell pellet was depleted of erythrocytes by two treatment steps with 50 ml erythrocyte lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA pH 7.3). After washing with PBS, cells were incubated for 10 min on ice with FACS buffer (1 % fetal bovine serum in PBS) containing 1 μg of purified anti-mouse CD16/CD32 Fc block (eBioscience, Frankfurt am Main, Germany) per 10⁶ cells. Cells were subsequently stained for 30 min at 4 °C in the dark with CD45-APC-Cy7, GR1-BV510 and Ly6C-PerCP-Cy5.5 (all from BioLegend) and then fixed for 10 min at room temperature with 1 % paraformaldehyde (PFA; Merck, Darmstadt, Germany) in PBS. Samples were analyzed using FACS Canto II flow cytometer and FACS Diva software (BD Bioscience, Heidelberg, Germany).