Rmflt3 l

Bone marrow was isolated from or WT C57BL/6J mice for generation of iDCs^{57 (link),58 (link)}. Following erythrocyte lysis, the bone marrow cells were resuspended in complete DC medium (RMPI 1640 + 25 mM HEPES + 10% FBS, 1% l-glutamine, 1% 200 mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin–streptomycin, 0.5% sodium bicarbonate, 0.01% 55 mM 2-mercaptoethanol) supplemented with rmGM-CSF (50 ng/mL) and rmFlt3-L (200 ng/mL) (Biolegend, San Diego, CA). The culture medium was changed on day 9 of culture. After 16 days of culturing, non-adherent cells and loosely adherent cells were harvested and gently washed by DPBS for subsequent labeling experiments or for T-cell co-culture experiments. In the iDC-OT-I CD8+ T-cell interactions experiments, iDCs were activated overnight with class C ODN 2395 (InvivoGen, San Diego, CA) and then cultured with or without indicated antigen peptides at 37 °C for 30 min. Non-adherent cells in the culture supernatant and loosely adherent cells were harvested and gently washed with DPBS for subsequent experiments. iDCs were cultured with or without indicated antigen peptides or tumor lysates (tumor cell/DC ratio = 10:1) and with indicated T cells (T cell/iDC ratio 1:2) in a complete T-cell medium prior to endpoint analysis as described below.

BM cells were isolated from femur and tibia of wt, Siglec-H ko, Hck ko or Hck/Fgr/Lyn ko mice and erythrocyte lysis was performed with 1 x ACK solution. BM cells were then cultured for 8 days at a concentration of 2 x 10⁶ cells/ml in complete RPMI medium [4 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 50 µM 2-mercaptoethanol, 100µM nonessential amino acids, 1 mM sodium pyruvate, and 10% FCS with 50 ng/ml rmFlt3L (Biolegend)] in 25-cm² cell culture flasks (each with 5 ml of cell suspension). On d4 of culture half of medium per flask was replaced by fresh medium containing 50 ng/ml rmFlt3L. After d8, CD11c⁺PDCA⁺ pDCs were purified with magnetic-activated cell-sorting separation columns using mouse pDC isolation Kit (Miltenyi Biotec).