Wright giemsa stain

After the experiment, the BALF was prepared based on the previous study [20 (link)]. BALF was centrifuged at 1,500 g for 15 min at 4°C, and the supernatant was collected to obtain the infiltrated cells. Then, the sediment cells were resuspended with ice 0.9% saline and stained with Wright-Giemsa stain (Jiancheng Bioengineering Institute, China). Cells were counted using a hemocytometer under a microscope. The inflammatory cytokine (TNF-α, IL-6, and IL-1β) levels in BALF were measured using a commercial kit per the manufacturer's protocols (Jiancheng Bioengineering Institute, China).

Total of 300 cells were seeded into 60-mm dishes. After 12 days, the culture medium was discarded, and each dish was washed twice with PBS. Then, the cells were stained with Wright-Giemsa stain (Nanjing Jiancheng Bioengineering) according to the attached instructions. Colonies were imaged and counted under the microscope.

Recovered BAL fluid was centrifuged at 800 rpm for 10 minutes at 4 °C, and the pellets were resuspended and seeded at a density of 1 × 10⁵ mononuclear cells per well in 6-well cell culture plate. The cells were maintained in medium consisting of DMEM/F12 supplemented with 10% fetal bovine serum (Invitrogen), 2 mmol/l L-glutamine (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen), and 2.5 ug/ml amphotericin B (Invitrogen) and incubated at 37 °C in 5% CO₂/95% air. Medium was changed first after 24 hours and then every 3 days. Single separated fibroblastoid colonies termed CFU-Fs were identified 14 days after initial plating. For staining and enumeration of CFU-Fs, adherent CFU-Fs colonies were fixed with 10% formaldehyde and stained with Wright-Giemsa stain (Jiancheng, Nanjing, China). To study mesenchymal cells obtained from an individual rat, all colonies growing in 6-well cell culture plate were trypsinized and passed into a 100-mm plate. A homogeneous population of mesenchymal cells was obtained from individual rats after 3–5 passages.