Bryte hs flow cytometer

MG63 cells were seeded at 1 × 10⁴ cell/cm² in 56 cm² Petri dishes, incubated 24 h to allow cell adhesion, and treated with the selected compounds at their IC₅₀ values. Doxorubicin and methotrexate were tested at 0.5 and 0.1 µM, respectively. After 24 h of treatment, cells were detached, counted, and stained according to the protocol reported by Erba et al. [29 (link)], with minor adjustments. Briefly, 1 × 10⁶ cells were pelleted and resuspended in trisodium citrate 0.1% w/v (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany), RNase 10 μg/mL (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany), IGEPAL^® CA-630 0.01% v/v (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany), and 50 μg/mL propidium iodide (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany). After 30 min of incubation at 37 °C in the dark, cells were analyzed through a BRYTE HS Flow Cytometer (Bio-Rad, Hercules, CA, USA) equipped with a Xe/Hg lamp tuned at 480 nm. PI fluorescence was collected with an emission band centered at 600 nm, on a linear scale. DNA distribution in the cell cycle was analyzed by the MODFIT software (Verity).

Human adipose-derived MSCs were detached with 0.1% trypsin, 0.02% EDTA, washed twice in DPBS and centrifuged. The pellet was suspended in 0.01% nonidet P-40, 10 mg/mL RNase, 0.1% sodium citrate and 50 μg/mL propidium iodide (PI), for 30 min at room temperature in the dark. Propidium iodide fluorescence was analyzed using a Bryte HS flow cytometer (Bio-Rad, Hercules, CA, USA) equipped with Hg lamp and analyzed with ModFit 4(Verity Software House, Topsham, ME, USA) software.