Red cell lysis buffer

Long bones were dissected from 4–6 month C57Bl/6 female mice. The epiphyses were removed from both femurs and tibias, and the bone marrow was flushed with 1× PBS. Bone marrow was treated with Red Cell Lysis Buffer (eBioscience, Inc., San Diego, CA, USA), and cells were plated in 10 cm dishes (Corning, Corning, NY, USA) with α-MEM supplemented with 10% (vol/vol) fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT, USA), and 25 ng mL⁻¹ macrophage colony-stimulating factor (M-CSF) for 24 hours. Cells were then plated into 24-well plates at 4 × 10⁵ per well with osteoclast differentiation media (100 ng mL⁻¹ RANKL and 25 ng mL⁻¹ macrophage colony-stimulating factor (M-CSF)) for 72 hours. Cells were lifted using 100 μL per well of 0.02% EDTA (Bio-rad Laboratories) and P100 pipette tips to scrape the bottom of each well. Cells were plated onto either bovine bone chips (Fisher; Immunodiagnostic Systems) or polymer films at 1 × 10⁵ per well in a 96 well plate (Fisher) in osteoclast differentiation media. 24 hours after plating onto bone or films, cells were given an additional media change with osteoclast differentiation media and stained and imaged 12–24 hours later.
All procedures involving animals were performed following a protocol approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC, PRO00009377).

Bone marrow-derived neutrophils (BMDNs) were isolated by using discontinuous Percoll gradients, as previously described.^{18 (link)} Briefly, following the surgical dissection of the bones, the femur and tibia were flushed with sterile PBS (Sangon, Shanghai, China) and passed through a 70 μm sterile filter (Fisher Scientific, Waltham, USA) to obtain the crude bone marrow cells. After centrifugation at 600 g for 5 min at 4°C with the brake off, cells were resuspended in 4 mL PBS and loaded on top of a discontinuous density gradient (62% and 81% Percoll) and centrifuged at 1500 g for 20 min at 4°C with the brake off. BMDNs were collected between 62% and 81% Percoll layers and suspended in 4 mL Red Cell Lysis Buffer (eBiosciences, multispecies) for 5 min on a rocker, washed with PBS, and centrifuged at 600 g for 5 min before resuspending in RPMI1640 (Hyclone, Logan, USA). Trypan blue dye exclusion indicated that cell viability was 95%–97% after the isolation. The purity of the isolated neutrophils was routinely 90%, as confirmed by flow cytometric analysis.

The spleen, thymus, and lymphoid node were pressed through a 200-gauge mesh. Splenic single-cell leukocyte suspensions were prepared by lysing the erythrocytes with red cell lysis buffer (Thermo Fisher Scientific). The CNS including the brain and spinal cord were removed and pressed through a 200-gauge mesh, and CNS mononuclear cells were then collected following 40% Percoll (GE Healthcare, no. 17089101) density gradient centrifugation.
For isolation of colon lymphocytes, the colon was cut and flushed with ice-cold phosphate-buffered saline (PBS) and then cut into 1-cm-long piece and incubated in extraction buffer (2% fetal bovine serum, 2 mM EDTA, and 1 mM dithiothreitol in PBS) with shaking (200 rpm) at 37°p for 30 min. After extensive washing to remove intraepithelial lymphocytes, the lamina propria was minced and incubated at 37° with shaking (200 rpm) for 60 min in digestion buffer [RPMI with 2% fetal bovine serum, type II collagenase (1 mg/ml), and dispase (0.5 mg/ml)]. The supernatant was collected, and lymphocytes were purified by Percoll gradient centrifugation.