Rbc lysis buffer

BM was collected from femurs and tibias of 4-week-old mice and suspended in α-MEM media (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma-Aldrich) and 100 units/mL penicillin/streptomycin (Thermo Fisher Scientific), passed through a 40 μm cell strainer and treated with RBC lysis buffer containing ammonium chloride (Cell Signaling Technologies, 46232). BM cells were plated overnight in α-MEM in a T175 flask. Nonadherent cells were collected, and 2.5 × 10⁵ cells were plated in each well of a 24-well plate in α-MEM supplemented with 100 ng/mL murine macrophage CSF (M-CSF; PeproTech, 315-02) for 3 days. For generation of BM-derived osteoclasts, cells were further incubated in α-MEM with 100 ng/mL M-CSF and 100 ng/mL recombinant murine soluble receptor activator of NF-κB ligand (RANKL; PeproTech, 315-11C) for an additional 4 days (media changed on days 2 and 3), and the presence of multinucleated giant cells was confirmed by light microscopy. For generation of BM-derived macrophages, RANKL was omitted from cell cultures.

Lymphocytes were taken from the spleen and lymph nodes of C57BL/6, OT-1, and STAT1−/− mice with or without FLT3L treatment. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in 0.5% BSA/PBS (FACS buffer). Cells were stained with PE-conjugated CD44 antibodies in the staining buffer, washed, and resuspended in the staining buffer. To assess the functions of CD44high CD8 T cells, CD44-positive cells were isolated using the EasySep™ PE Positive Selection Kit II (Stem Cell Technologies). The isolation efficiency was confirmed by flow cytometry analysis. For the T cell proliferation assay, lymphocytes (1 × 10⁶ cells/ml) were suspended in phosphate-buffered saline (PBS) and labeled with the CellTrace Violet™ (Thermo Fisher Scientific, Waltham, MA) at a concentration of 10 μM at 37 °C for 20 min. After labeling, an excess of 10% RPMI/FBS was added to the samples to quench the reaction. Following centrifugation and extensive washing, cells were resuspended in 200 ml of 10% RPMI/FBS with T cell stimulant: either α-CD3 (1.25 ug/ml) and α-CD28 (0.25 ug/ml) antibodies for lymphocytes from C57BL/6 mice, or OVA257-264 (SIINFEKL, 10 or 50 ng/ml) peptides for lymphocytes from OT-1 mice for 24 to 48 h. Cells were then harvested for analysis by flow cytometry.

Lymphocytes were collected from the spleen and lymph nodes of C57BL/6 mice with or without administration of FLT3L treatment, and single-cell suspensions were prepared. RBCs were lysed by the addition of excess RBC lysis buffer twice (Cell Signaling Technology, Danvers, Massachusetts) followed by extensive washing in 0.5% BSA/PBS (FACS buffer). Cells were stained with fluorescence-conjugated CD3, CD8, and CD44 antibodies in the staining buffer, washed, and then resuspended in the staining buffer. The CD44^high CD8 T cells were sorted by flow cytometry for adoptive transfer via retro-orbital injection.