Alexa 488 anti cd8α clone 53 6.7

Splenocytes were aliquoted into FACS tubes (6 × 10⁵ /tube) and centrifuged at 200 × g for 10 min. Cell pellets were resuspended in 80 µl FACs buffer (PBS buffer with 2% BSA and 0.10% sodium azide) and 20 µl of mouse serum. After 10 min incubation at RT, the cells were stained with antibodies for 30 min in the dark at 4°C. The cells were washed twice with FACS buffer and analyzed on a LSRII cytometer using Flow-Jo software (Tree Star). Antibodies used were Alexa-488 anti-NK1.1 (clone P136), Alexa-488 anti-CD45R (B220) (clone RA3–6B2), Alexa-448 anti-CD4 (clone GK1.5), Alexa-488 anti-CD8α (clone 53–6.7), Bv421 anti-CD11b (clone M1/70), BV605 anti-Ly6C (clone HK1.4), APC anti-Ly6G (clone1A8), PE anti-Ly6G (clone 1A8), Alexa 700 anti-F4/80 ( clone BM8), BV421 Rat IgGb, BV605 Rat IG2b, APC Rat IgG2a, and PE Rat IgG2a, Alexa700 Rat IgG2a (obtained from BioLegends); APC anti-SIGNR1 (clone 22D1) and APC hamster IgG (obtained from eBioscience). Gating strategy for identification of myeloid spleen populations is outlined in Suppl. Fig. 1. The number of each myeloid population/spleen was calculated from the % of the population determined by flow cytometry and the total spleen cell counts.