Easysep mouse cd11b positive selection kit

Primary bone marrow monocytes were isolated from the bone marrow of femur and tibiae of four-week-old C57BL/six female mice [51 (link)]. Mice were euthanized and femurs and tibiae were harvested and cleaned free of soft tissue. Bone ends were removed, and bone marrow was flushed using αMEM (Gibco) plus 1% 100 U/mL penicillin/100 mg/mL streptomycin (Gibco). A total of 10 femurs and 10 tibiae were flushed. Bone marrow aspirate was plated in osteoblast growth medium (αMEM (Gibco) supplemented with 10% FBS (HyClone) and 1% 100 U/mL penicillin/100 mg/mL streptomycin (Gibco)) for 24 h.
After 24 h, nonadherent cells were harvested by collecting the medium and centrifuging for 10 min at 1000 rpm. Cells were resuspended in 2 mL of 1X PBS plus 5% FBS (HyClone) for separation, as previously described [51 (link)]. CD11b+ mononuclear cells were collected using the EasySep™ Mouse CD11b Positive Selection Kit (STEMCELL Technologies, Vancouver, Canada). CD11b+ cells were maintained in a medium of αMEM (Gibco), 10% FBS (HyClone), and 1% 100 U/mL penicillin/100 mg/mL streptomycin (Gibco) plus 10 ng/mL M-CSF (PeproTech, Rocky Hill, NJ, USA) [51 (link)]. These CD11b+ cells were used as a source of enriched osteoclast precursors.

Dissected orthotopic pancreatic tumors were collected following mice sacrifice on day 20 from tumor inoculation for analysis of tumor infiltrating lymphocytes (TILs). Each tumor was digested by using a tumor dissociation kit and processed sequentially through 40-µm and 100-µm nylon filters and brought to a volume of 20 mL of CTL medium. Suspensions were centrifuged at 1,500 rpm for 5 min. Cell pellets were suspended in 4 mL of ammonium-chloride-potassium (ACK) lysis (Quality Biological) and subsequently spun at 1,500 rpm for 5 min. Cell pellets were then resuspended in 6 mL 80% Percoll (GE Healthcare LifeSciences), overlaid with 6 mL 40% Percoll and centrifuged at room temperature for 25 min at 3,200 rpm without brake. The lymphocyte layer was removed and quenched with 30 mL of CTL media. Isolated TILs were enriched for CD11b+ cells using the EasySep™ Mouse CD11b Positive Selection Kit (Stemcell Technologies).

Microglia were purified by immunopanning from postnatal day 1 (P1) forebrain and cultured as previously described [21 (link)]. Briefly, after removing the meninges, the brains were washed with PBS and then transferred to 0.25% trypsin-EDTA, followed by gentle agitation for 30 min. The complete media, DMEM/F12 (Gibco, 11320033, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM l-glutamine, 100 μM non-essential amino acids and 2 mM sodium pyruvate, were used to stop the trypsinization reaction. A single-cell suspension was obtained by trituration through pipetting. Cell debris and aggregates were removed by passing the single-cell suspension using a 100 μm nylon mesh. The prepared single-cell suspension was cultured in T175 flask for 13 days with complete media change on day 6. Microglia were separated using the EasySep Mouse CD11b Positive Selection kit (StemCell, #18970, Cambridge, MA, USA). The magnetically separated microglia-rich fraction was seeded on 6-well plates with complete media.