Cell steiner 70 μm filter

Femurs and tibia of C57BL/6 male mice were dissected, and the epiphyses were surgically removed with scissors to perform internal flushing with sterile PBS. The cells thus obtained from bone marrow were filtered using Cell Steiner 70 μm filter (Corning, USA), and the flow-through cells were twice centrifuged and washed at 300 g for 5 min. Subsequently, cells were lysed with 0.83% NH₄Cl (3 min/4°C) and cultured in DMEM high glucose medium (Gibco) containing 10% FBS (Gibco) and 30% of supernatant medium from L929 cells (ATCC, Cell Lines) (23 (link)). The culture medium was refreshed on day 4 and subsequently maintained until day 7 to promote BMDM differentiation. On day 7, 2 × 10⁵ Mϕs/cm² were polarized to M1 by 5 ng/mL IFN-γ (R&D Systems, USA) and 50 ng LPS/mL (E. coli-LPS, Sigma-Aldrich, USA) incubation for 24 h. In contrast, M2 polarization was induced by 10 ng IL-4/mL and 10 ng IL-13/mL (R&D Systems) for 24 h.

Bone marrow-derived macrophages (BMDM) were isolated from the femur and tibia of the control (FVB/N) and hypertensive (TGM123-FVBN) male mice. Cells were filtered using a Cell Steiner 70 μm filter (Corning, USA), and the flow-through cells were washed twice with PBS by centrifugation at 300 g for 5 min. Subsequently, cells were lysed with 0.83% NH₄Cl (3 min/4°C) and cultured in RPMI 1640 (Gibco) and DMEM High Glucose Medium (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin-streptomycin (Gibco), and murine M-CSF (macrophage colony-stimulating factor) (PeproTech, USA). The culture medium was refreshed on day 3 and maintained until day 7 to promote BMDM differentiation. On day 8, the cells were polarized to M1 by 10 μg/ml IFN-γ (R&D Systems) and 1 mg/ml LPS (E. coli-LPS, Sigma-Aldrich, USA) and to M2 by 10 μg/ml IL-4 (R&D Systems) and 10 μg/ml IL-13 (R&D Systems). After induction of polarization, cells were cultured for 48 h and thereafter used for the RNA extraction, one well per condition per animal.