Trypan blue staining method

In step eight, MSCs were divided into seven groups, which were exposed to hyaluronic acid from six commercial brands and phosphate-saline buffer – PBS (control group):

Group 1: AD-MSC in contact with Fermathron® (Hyaltech),

Group 2 AD-MSC in contact with Orthovisc® (J&J),

Group 3 AD-MSC in contact with Synvisc® (Sanofi),

Group 4 AD-MSC in contact with Synovium® (LCA Pharmaceutical),

Group 5 AD-MSC in contact with Suprahyal® (Zodiac),

Group 6 AD-MSC in contact with Osteonil® (TRB Chemedica),

Group 7 AD-MSC in contact with PBS (Thermo Fisher Scientific).

In the preparation process, the cells were detached from the culture bottle with TrypLE reagent (Thermo Scientific) and centrifuged at 300 × g for 5 min, counted in the Countess apparatus (Thermo Fisher), and resuspended in hyaluronic acid or PBS at the concentration of. After 4, 24, and 48 h of incubation at room temperature and atmosphere, cell viability analysis was performed through the Countess counter by the Trypan Blue staining method (Thermo Fisher Scientific), according to the supplier's recommendations. Two independent experiments were performed, each with three different AD-MSC strains (AT8, AT11, and AT13). For each time interval, a single viability reading was made, and the same experimental conditions were followed.

A549 cells were cultured in DMEM with 10% FBS and challenged with apoptosis insults H₂O₂ (5 mM) for 2 h or UV (1,000 mJ/cm²) for 2 h. For pyroptosis cells were treated with lipopolysaccharide (10 ng/mL) for 4 h then nigericin (10 μM) for 6 h. For induction of necroptosis cells were administered rPly (0.1 μg/mL) for 4 h or zVAD-fmk (20 μM) and cycloheximide (1 μg/ml) for 1 hour before addition of TNF (100 ng/ml) (Coats et al., 2005 (link)). Host cell cytotoxicity of recombinant PspA and Ply were evaluated by WST-1 solution according to the manufacturer’s recommendations (Roche, Germany). In other instances, cell viability was measured using trypan blue staining method according to the manufacturer’s instructions (ThermoFisher, USA).