0.83 m nh4cl buffer

Bone marrow-derived DCs (BMDCs) were isolated from the bone marrow cells of C57BL/6 (H-2^b) mice.26 (link) Briefly, bone marrow was collected from the tibias and femurs by flushing with RPMI 1640 using a 1 mL syringe with a 26 G needle (BD Biosciences, San Jose, CA, USA). After one wash (490×g, 5 minutes) in RPMI 1640 medium, the cells were incubated with 5 mL of 0.83 M NH₄Cl buffer (Sigma-Aldrich) to lyse red blood cells at 37°C for 5 minutes. Then, cells were centrifuged twice in RPMI 1640 medium at 150×g. The bone marrow cells (2×10⁶ cells) were collected and cultured in 100 mm petri dishes (BD, Franklin Lakes, NJ, USA) containing 10 mL of the RPMI 1640 medium supplemented with 10% of heat-inactivated fetal bovine serum, 50 IU/mL penicillin, 50 mg/mL streptomycin, and 20 ng/mL mouse recombinant granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA). After 7 days, nonadherent and loosely adherent cells (immature DCs) were harvested, washed, and used for in vitro experiments.

Femurs and tibiae were collected, and the muscle attachments were carefully removed. Intact bones were incubated in 70% (v/v) ethanol for 1 minute for disinfection and were then washed with phosphate-buffered saline (PBS; HyClone). Next, both ends of the bones were cut with scissors, and the marrow was flushed out with RPMI 1640 medium (Thermo Fisher Scientific) using a 1 mL syringe connected to a 26-gauge needle. Clusters within the marrow suspension were disintegrated via vigorous pipetting. After one wash (15,000 rpm, 5 minutes) in RPMI 1640 medium, the red blood cells were depleted using 0.83 M NH₄Cl buffer (Sigma-Aldrich). Bone marrow cells (2×10⁶ cells) were collected and cultured in 100 mm Petri dishes containing 10 mL of RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 50 IU/mL penicillin, 50 µg/mL streptomycin, and 20 ng/mL mouse recombinant granulocyte-macrophage colony-stimulating factor (R&D Systems, Inc., Minneapolis, MN, USA). After 7 days, the nonadherent and loosely adherent cells (imDCs) were harvested, washed, and used for in vitro and in vivo experiments.

Both the femurs and tibiae were collected, and the muscle attachments were carefully removed. Intact bones were soaked in 70% (v/v) ethanol for 1 minute for disinfection and then washed with PBS. Next, both ends of the bones were cut with scissors, and the marrow was flushed with Roswell Park Memorial Institute (RPMI) 1640 medium using a 1-mL syringe with a 26-G needle. Clusters within the marrow suspension were disintegrated by vigorous pipetting. After one wash (490× g, 5 minutes) in RPMI 1640 medium, the red blood cells were depleted with 0.83 M NH₄Cl buffer (Sigma-Aldrich). Bone marrow cells (2×10⁶ cells) were collected and cultured in 100-mm Petri dishes containing 10 mL of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 IU mL⁻¹ penicillin, 50 mg mL⁻¹ streptomycin, and 20 ng mL⁻¹ mouse recombinant granulocyte macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA). Bone marrow cells (5×10⁵ cells) were collected and cultured in 100-mm Petri dishes containing 10 mL DMEM supplemented with 20% heat-inactivated fetal bovine serum, 50 IU mL⁻¹ penicillin, 50 mg mL⁻¹ streptomycin, and 20 ng mL⁻¹ recombinant murine macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA). After 7 days, nonadherent and loosely adherent cells (BMDCs) or adherent cells (BMDMs) were harvested, washed, and used for in vitro experiments.