Antibiotic antimycotic solution ab am

Jurkat (TIB-152) human T cell lymphoma and THP-1 (TIB202) human acute monocytic lymphoma cell lines were obtained from ATCC (Manassas, VA). The BV-2 murine microglia cell line was a generous gift of Prof. Rosario Donato (Università degli Studi di Perugia, Perugia, Italy) [9 (link)]. MH-S murine alveolar macrophage cell line originally obtained from ATCC was kindly provided by Dr. Dolores Solis (Madrid, Spain). The cell lines were cultured in RPMI medium containing 10% (v/v) fetal bovine serum (FBS), 2 mM glutamine, and 1% Antibiotic Antimycotic Solution (Ab/AM) (all from Sigma-Aldrich, St Louis, MO), at 37°C in 5% CO₂/air. The cell lines were regularly tested for Mycoplasma contamination by fluorescence microscopy using DAPI staining (Molecular Probes Life Technologies, Carlsbad, CA).

Parenchymal lung tissue from explants or non-tumorous lung cancer resections was dissected out, avoiding visible airways. Tissue pieces (< 30 mm³) were washed with Dulbecco’s phosphate-buffered saline (DPBS) and cut into small pieces (< 3 mm³) before being enzymatically digested using 100 U/mL DNase I (Sigma-Aldrich), 300 U/mL collagenase type 1 (Gibco) and 1 mg/mL hyaluronidase (Serva Electrophoresis) in DPBS for 1.5–2 h at 37 °C. Single-cell suspensions were separated from digested tissue by filtration through a 100 µm filter. Single-cell suspensions were incubated in red blood cell (RBC) lysis buffer (155 mM NH₄CL, 10 mM KHCO₃) for 5 min to lyse RBCs. An aliquot of the isolated primary lung cells was plated for colony forming unit-fibroblast (CFU-f) assay as described below and the remaining cells were immediately processed for flow cytometry or cryopreserved in StemMACS MSC Expansion media (Miltenyi Biotec) containing 1% antibiotic–antimycotic (AB/AM) solution (Sigma-Aldrich) mixed 1:1 (v/v) with DPBS containing 15% DMSO, 50% FBS (Life Technologies) and 20 IU/mL Heparin (Leo Pharma).