Flow cytometry

Spleen was removed in a 35 mm petri plate with 5 ml RPMI 1640 and digested mechanically and passed through 70 μm filter screen.

The cell suspension was centrifuged, and the pellet was incubated in RBC lysis buffer. The resulting cell suspension was washed in 1xPBS and blocked with Fc Block (Biolegend, Cat# 101302, 1 μl/50 μl).

The cells were incubated with MC1R (Invitrogen, Cat# PIPA521911, 1.39 µg) antibody followed by fluorophore conjugated primary antibodies for extracellular markers (Biolegend Cat# 101235,

CD11b-BV421(0.25 µg); Cat# 127641, Ly6G-BV-650 (0.25 µg); Cat# 128041, Ly6C-BV785 (0.125 µg); Cat# 100516, CD4-APC (0.25 µg); Cat# 100751, CD8a-BV510 (0.5 µg); Cat# 152405, CD19-PerCP-Cy5.5 (0.25 µg); Cat# 102036, CD25-BV605 (0.3 µg)) and AF488 (Invitrogen, Cat# A11034, 1:200).

Zombie dye (Biolegend, Cat# 423101, 1µl/sample) was used to differentiate between live and dead cells. Helper T cells and cytotoxic T cells were identified by CD4+ and CD8+, respectively.

CD4+CD25+ cells were used to mark Tregs. CD19+ cells were used as marker for B cells. Monocytes were identified as CD11b+Ly6G-Ly6^highh cells and neutrophils were marked by CD11b+Ly6C-Ly6G+. For monocytes and neutrophils, CD11b-positive cells were first extracted from the live cell subset by

expansion with SSC-A, followed by Ly6C. After expansion with Ly6C and Ly6G, we excluded Ly6G-positive cells. Monocytes were identified as CD11b+Ly6G-Ly6C high cells, and neutrophils were marked by CD11b+Ly6C-Ly6G+.

SORP 5 Laser BD Fortessa X-20 (BD Bioscience) and FlowJo v10.7.1 (Becton Dickson & Company) software was used for data acquisition.