Cytocentrifugation

TK6 cells (CRL-8015™, purchased American Type Culture Collection, ATCC, Manassas, VA) were cultured in RPMI-1640 medium supplemented with 2 mM L-Glutamine, 1 mM Sodium pyruvate, 4.5 g/L Glucose, 10 mM HEPES, 1.5 g/L NaHCO₃ (PAN™ Biotech, Aidenbach, Germany), 10% horse serum (Life Technologies; Thermo Fisher Scientific, Waltham, MA) and incubated at 37 °C, 5% CO₂/95% air and 98% relative humidity. Cells were passaged until sufficient numbers allowed for experimental purposes, counted, and seeded (2 × 10⁵ cells/well) into 12-well plates (Falcon^®, Corning, Tewksbury, MA, 3.8 cm²/well). For the non-genotoxin vs genotoxin assay, TK6 cells were seeded (2 × 10⁵ cells/well) in 24-well plates (CELLSTAR^®, Grenier Bio-One, 1.9 cm²/well). The cells were subsequently fixed in Carnoy’s solution or processed via cytocentrifugation (Thermo Fisher Scientific, Waltham, MA). One thousand cells had been scored per treated sample for MN induction. The TK6 MN data were generated from independent duplicates. Each experiment consisted of a single sample per dose.

The number of PMNs and WBCs in BALF was assessed using a Diff-Quik staining assay according to the described procedure before [27 (link)]. Briefly, the BALF was centrifuged at 3000 rpm for 15 min and resuspended with 100 μL cold PBS. And the number of cells was counted using a hemocytometer. And the smears were made using cytocentrifugation (Thermo Fisher Scientific, USA) and visualized using Diff-Quik staining kit (Sysmex Co., Japan). At least 200 cells on each slide were calculated under a microscope according to the stained phenotype of macrophages, neutrophils, lymphocytes, and eosinophils.