Pen strep glutamate

AMs were obtained from BAL as described previously [35 (link)]. C57BL/6 mice were sacrificed, and alveolar lavage was performed by flushing the airway for five times with 1 mL cold, sterile BAL washes via a tracheal incision. Alveolar lavages were centrifuged at 4 °C, 800× g for 5 min, and cells were obtained and purified to culture plates by adherence in complete medium (RPMI-1640 with 10% FBS and 1% Pen/Strep/glutamate, Thermo Fisher Scientific, MA, USA); 1 × 10⁵ cells per well of co-cultured and mono-cultured AMs were seeded in non-treated 24-well plate for 2 h at 37 °C in 5% CO₂ cell incubators. The non-adherent cells were washed off with warm PBS and cultured with a complete medium.
For co-culture of AMs with BAL–, BM– or PR8–neutrophils, BAL-, BM- or PR8-neutrophils were plated in a non-treated 24-well plate with AMs (co-cultured at a ratio of 1:1, 2:1, and 8:1). Then the cells were cultured in complete medium containing 10 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, BioLegend, San Diego, CA, USA) after 24 h. Subsequently, the cells were analyzed by flow cytometry or bulk RNA-seq.

C57BL/6 mice were sacrificed, and the BM cells were collected from tibia and femur through a 25-gage needle by flushing with cold, sterile PBS. Cells were passed through a 70 μm cell filter in a 50 mL falcon tube and pelleted at 800× g for 5 min, and the pellet was suspended in ACK lysis buffer at room temperature (RT). After centrifugation at 800× g for 5 min, BM cells were resuspended and cultured with a complete medium (DMEM with 10% FBS and 1% Pen/Strep/glutamate, Thermo Fisher Scientific) supplemented with 50 ng/mL recombinant murine macrophage colony-stimulating factor (M-CSF, BioLegend) to induce differentiation into a macrophage phenotype. After 7 days, BMDMs were generated for subsequent experimentation. For co-culture of BMDMs with BAL– or BM–neutrophils, following replating of BMDMs, 1 × 10⁵ cells per well of co-cultured and mono-cultured BMDMs were plated in a 24-well plate at 37 °C and 5% CO₂ cell incubators.