Trypan blue staining solution

HCCLM3 cells which had been treated with 1, 10, 100 nmol/L DEX for 24 h were collected. HCCLM3 single-cell suspension (100 μL) and 100 μL trypan blue staining solution (Sigma–Aldrich, CA, USA) were reacted for 3 min. The dead cells were counted and recorded by a CountStar cell counter, and the cell viability was calculated.

The isolation of PEMs was performed as described by Zhang et al. (2008) (link) with some modifications. Eight to twelve-week-old female BALB/c mice were injected via an intraperitoneal route with 1 mL of 3% (w/v) Brewer thioglycolate (Himedia, India) solution in PBS. PEMs were harvested after 48 h by peritoneal lavage with 2% hiFBS-RPMI1640 ice-cold medium (GE Healthcare Life Science-HyClone, South Logan, UT, United States) containing 1% penicillin–streptomycin (PenStrep; Sigma, United Kingdom). PEMs were collected by centrifugation (×500 g, 4°C, 10 min) and then resuspended in RPMI medium containing 10% (v/v) hiFBS. The viability of PEMs was estimated using trypan blue staining solution (Sigma-Aldrich, St Louis, MO, United States) in an improved Neubauer chamber (Precicolor, HBG, Germany) under light microscopy.

Cardiomyocytes were seeded at 30,000 cells/well concentration the day before the assay.
The next day the same treatments carried out in the HepG2 cells were performed, but with an incubation time of 2 h, due to the higher sensitivity of the primary cells to H₂O₂ compared to the immortalized HepG2 cell line. The evaluation of cell survival was carried out, first using a Trypan blue Staining Solution (Trypan blue 1:2, Sigma-Aldrich, St. Louis, MO, USA). The number of vital cells was measured with the Biorad TC20 tool™. The mortality rate induced by H₂O₂ was calculated vs. untreated (CTR) and the protective activity of EVs vs. H₂O₂ was also calculated.
A second assay was carried out to quickly discriminate the live cells from the dead cells by simultaneously staining with green-fluorescent calcein-AM to estimate intracellular esterase activity, and red-fluorescent ethidium homodimer-1 was used to indicate the loss of plasma membrane integrity (live and dead assay). The fluorescence was detected using a fluorescent microscopy Axiovert200 with Apotome2.1 (Zeiss, Oberkochen, Germany).

The proliferation rates of the MSC and chondrocyte cell lines were calculated following printing using a trypan blue exclusion test. Cells were either printed or manually pipetted at a concentration of 10⁶ cells per mL into the wells of a six-well plate containing cell culture media. At each respective time point, cells were trypsinised and a 10 μL aliquot combined with an equal volume of trypan blue staining solution (Sigma-Aldrich) prior to counting using a haemocytometer.