Countess cell counting chamber slide

SH-SY5Y were differentiated and treated with the extracts (50 μg mL⁻¹) and after 24 h cells were stained with 0.4% trypan blue. The viability was evaluated in Countess™ Cell Counting Chamber Slides (Invitrogen, Carlsbad, CA, USA) using the Countess® Automated Cell Counter (Invitrogen). The number of the cells was determined for each sample, and dead cells were discriminated by the incorporation of trypan blue. Percent of viability was calculated as follows:
Viability (%) = ( Live cell number/Total cell number) × 100.

Cisplatin-resistant HCC cells (HCC-LM3/DDP and Huh-7/DDP) and HCC cells (HCC-LM3 and Huh-7) were in the logarithmic growth stage and digested with trypsin. Cell number was calculated using Countess™ Cell Counting Chamber Slides (Catalog number: C10228, Invitrogen™). With the cell density adjusted to 2 × 10⁴/mL, 100 μL medium was added to 96-well plates. Cisplatin at different concentrations was administered to treat the cells for 24 h, and the 96-well plates were further cultured in an incubator. A 10 μL CCK8 solution (Beyotime Biotechnology, Shanghai, China) was added to each well after 24, 48, or 96 h to incubate the plates for another 1 h in the incubator. As the culture ended, a microplate reader in which the 96-well plates were placed was exploited to check the absorbance (OD value) of each well at 450 nm, after which the determination was taken at the 24th, 48th, 72nd, and 96th hours.

An MTT 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (ThermoFisher Scientific, Grand Island, NY) assay was used to test the effects of PM₁₀ on HCET viability as reported before (Somayajulu et al., 2023 (link)). Briefly, 15,000 HCET cells were seeded in a 96 well plate, treated with 0, 25, 50, 100, 200, 500, 800 and 1200µg/mL PM₁₀ for 24h or PM₁₀ (100µg/ml) ± 50nM SKQ1 ± strain 19660 as described above in “Tissue culture and treatments”. At the end of the treatment, 5mg/ml MTT reagent was added to each well and incubated at 37°C for 4h and media removed. Dimethyl sulfoxide (DMSO, 100%) was added (50µl/well) and optical density was read at 540nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Data are shown as % cell viability + SD. To confirm the MTT data, a trypan blue exclusion cell viability assay was also performed using Countess cell counting chamber slides (Invitrogen, Carlsbad, CA) per the manufacturer’s protocol. Briefly, cells were treated as mentioned in “Tissue culture and treatments” and trypsinized 24h post treatment, centrifuged, resuspended in KSF media and incubated 1:1 with trypan blue. A 10μl aliquot was placed on the chamber slide and viable cells were counted using a Countess automated cell counter (Invitrogen, Carlsbad, CA). Data are shown as % cell viability + SD.

Hepatic mononuclear populations were isolated as previously described (Montes de Oca et al., 2016a (link); Stanley et al., 2008 (link)). Briefly, mice were sacrificed by CO₂ asphyxiation after which the liver was perfused by injecting 5-10 mL of 1x PBS through the portal vein. The liver was excised, weight and collected into 10 mL 2%FBS.PBS. Livers were passed through a 200 μm metal mesh held inside a tea strainer using the back of a 5 cc/ml syringe plunger (Terumo Medical). Mesh was washed with 10-15 mL 2%FBS.PBS and cell suspension was made up to 40 mL and centrifuged at 350 g in an Eppendorf Centrifuge 5810 R (Thermo Fisher Scientific). Hepatocytes were separated form leucocytes using a 33% (v/v) Percoll Density Gradient Media (GE Healthcare, Little Chalfond, UK) and centrifuged at 575 g for 15 minutes at RT with the brake off. The leucocyte cell pellet was incubated in 1 mL Red Blood Cell Lysis Buffer Hybri-Max (Sigma-Aldrich) for 6 minutes at RT. Cells were washed again in 10 mL 2%FBS.PBS after which cell pellet was diluted in DPBS (GIBCO) and Trypan Blue Stain (Invitrogen) and counted using Countess Cell Counting Chamber Slides on the Countess II FL (both from Invitrogen), as per manufacturer’s protocol.