Isolation and culture of primary macrophage (bone marrow derived macrophage, BMDM)

BMDM was isolated from mice bones and cultured as described earlier^65,71. Wild-type (C57BL6/J) mice were purchased from Jackson Laboratory. All mice are maintained in a specific pathogen free (SPF) facility at UT Southwestern Medical center. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the IACUC guidelines, the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines. For the isolation of BMDMs, femur and tibia were collected from mouse legs. Using 25G needle bone marrows were flushed out with Iscove's Modified Dulbecco's Medium (IMDM), (12440061, Life technologies) and processed for single cell suspension by passing through 22G needle two times. The suspension was centrifuged at 1000 RPM for 5 min. The pellet was re-suspended with BMDM culture media (L-cell-conditioned IMDM medium supplemented with 15% L929 supernatant, 10% FBS, 1% nonessential amino acid, and 1% penicillin–streptomycin) followed by seeding in three 150 mm culture dishes and cultured for 6 days to differentiate into macrophages, while at day 3, 10 mL fresh BMDM culture media was added into each plate. After day 6, the culture plate was washed with ice cold PBS and using cell scrapper cells were gently scraped with ice cold PBS. The BMDM was centrifuged at 1000 rpm for 5 min and re-suspended into BMDM media. The BMDM was counted and seeded in 6-well (2.5 × 10⁶/well) cell culture plates. After overnight incubation the BMDM was treated with LPS or HOTAIR siRNA and processed for further experiments.