BMM surface staining

At 7 days post-harvest, BMM were stained for surface markers of differentiation and assayed by flow cytometry. Medium was aspirated, and cells were washed in PBS, incubated in FACS buffer (FB; 5 mM EDTA and 0.1% BSA in PBS) (4 °C, >10 min), detached by tapping dishes firmly and pipetting in FB, aliquoted as 2.5 × 10⁵ cells per FACS tube, and pelleted by centrifugation (400 × g, 5 min). Supernatant was decanted, paraformaldehyde (PFA; 4% in PBS, 30 μl) was added, and tubes were flicked to mix and incubated (4 °C, 20 min). 1 ml FB was added, tubes were flicked to mix and centrifuged, and supernatant was decanted; this step was performed a total of three times. To block, normal mouse serum (Sigma #M5905, 10 μl) was added, and tubes were flicked to mix and incubated (room temperature—approximately 22 °C, 15 min). Staining was conducted with primary conjugated antibodies (BD Biosciences): PE rat anti-CD11b (#553311, 0.04 μg) and Alexa Fluor 647 rat anti-mouse F4/80 (#565854, 0.04 μg). Isotype controls were PE rat IgG2b, κ isotype control (#553989, 0.04 μg) and Alexa Fluor 647 rat IgG2a, κ isotype control (#557690, 0.04 μg). Compensation control samples were prepared using anti-surface marker antibodies separately, and a no-antibody control sample was prepared. Tubes were flicked to mix and incubated (4 °C, 1 h). 1 ml FB was added, tubes were flicked to mix and centrifuged, and supernatant was decanted; this step was performed a total of three times. Several drops of FB were added, and tubes were covered in foil and kept on ice until flow cytometry.