Automacs magnetic cell separator

BM was extracted from femur, pelvic bone, and tibias via crushing. CKIT⁺ cells were isolated via magnetic enrichment using anti-CD117 microbeads (Miltenyi Biotec, Carlsbad, CA) and an autoMACs magnetic cell separator (Miltenyi Biotec). Cells were then stained with anti-SCA1 PerCPCy5.5 (E13-161.7) and anti-CKIT APCe780 (2B8) to facilitate isolation of LSK cells by cell sorting on FACSAria III (BD Biosciences).

Cells were isolated from femurs and tibias of age-matched female donors. Bone marrow cells were subjected to T and B cell depletion by AutoMACS Magnetic Cell Separator (Miltenyi Biotec). Briefly, cells were incubated with anti-CD90.2 and anti-CD19 Miltenyi Biotec microbeads at 1:9 dilutions in AutoMACS buffer solution for 15 min at 4°C. Cells were then washed with AutoMACS buffer, resuspended at concentration of 10⁸ cells per 500 μl, and passed through AutoMACS Magnetic Cell Separator columns using the ‘Deplete’ program. Collected cells were washed and resuspended at the concentration of 1.5 × 10⁷ cells per 0.2 ml per mouse in lactated ringer’s solution (Abbott Laboratories).

Peyer’s patches (PPs) were digested for 40 min at room temperature with collagenase/DNase as previously described.⁵⁵ (link) CD11c⁺ cells were enriched using anti-CD11c microbeads and an autoMACS magnetic cell separator according to manufacturer’s instructions (Miltenyi Biotec, France).
After removal of adherent adipose tissue and resection of PPs, 14 cm of the SI was cut into representative pieces of each part of the SI (duodenum-jejunum and ileum), opened longitudinally, washed in cold PBS 1X and placed in complete medium (RPMI 5% FCS, 1% Penicillin/Streptamycin, 1% L-Glutamine, 10 mM HEPES and 50 μM β-mercaptoethanol) containing 1 mM dithiothreitol. SI was cut into 2 mm pieces, washed in cold HBSS (Thermo Fischer) containing 2% FCS and filtered with a 100 μm cell strainer. Epithelial cells were removed by incubating tissues in HBSS+2 mM EDTA buffer at 37°C for 20 min at 180 rpm. This step was repeated twice before digestion at 37°C in complete medium with collagenase VIII (1 mg/mL, Sigma) and DNAse I (200 μg/mL, Sigma) for 15 min at 180 rpm. Digestion was stopped by adding 20 mL of cold PEF 2% buffer (PBS1X + 5 mM EDTA +2% FCS). Debris were removed by filtration. The cells were washed with cold PEF 2% before staining.