96 well cell culture plate

CCDC18Co cells and CAFs were grown in DMEM in standard conditions, and CM from HT29 cancer cells were obtained as described above. For the experiments, CCD18Co cells and CAFs were seeded on day 1 at 4000 cells/well in 96-well cell culture plates (catalog no. 83.3924.005, Sarstedt, Nümbrecht, Germany) with DMEM and 10% FBS. Twenty-four hours later, medium was removed and cells were washed once with PBS. Then, 150 μL of CM/DMEM mix was added to CCD18Co cells. The mix contained 50% of fresh DMEM and 50% of CM from HT29 cells starved for 48h. To this mix, 1% FBS (final concentration) was added. Next, antibodies were added (10μg/mL for CCD18Co cells and 100μg/mL for CAFs). Then, cells were incubated at 37ºC and 5% CO₂ for 120h. Afterwards, cell viability was assessed using 3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) (MTT) staining (catalog no. M5655, Sigma-Aldrich). Absorbance was measured at 540 nm. Statistical analysis was performed using the t-test.

The culture medium for each type of MSC was removed and replaced with fresh medium, and 72 h later the medium containing the products released by each type of MSC were harvested as previously reported^{24 (link),25 (link)}. Briefly, the medium was centrifuged for 10 min to eliminate all cellular debris and apoptotic bodies, followed by filtering through a sterile filter with a pore diameter of 0.22 µm (Sarstedt, Nümbrecht, Germany). Secretomes obtained from the three cultures of each type of MSC were pooled together in a single secretome suspension. Secretomes obtained from hADSC, hDPSC and hWJSC were called hADSC-s, hDPSC-s, and hWJSC-s, respectively.
To evaluate the effects of each secretome on HSEC, these cells were cultured in 96-well cell culture plates (Sarstedt) at a cell density of 1.7 × 10⁴ cells/cm² with epithelial cell culture medium. 48 h later, the epithelial cell medium was removed, and cells were washed in phosphate-buffered saline (PBS) and cultured with epithelial cell medium supplemented with increasing concentrations of each type of secretome: 0%, 25% (500 µg of total protein per mL), 50% (1000 µg/mL), 75% (1500 µg/mL), 100% (2000 µg/mL). HSEC were cultured with each type and each concentration of secretome for 24, 48, 72 and 120 h.

Pure dLG-HG and genipin/dLG-HG and DMSO/dLG-HG dilutions were distributed into 96-well cell culture plates (Sarstedt) with 60-µL/well and allowed to crosslink overnight. LG-EpCs, HUVECs (5 × 10⁴/well), or LG-MSCs (2.5 × 10⁴/well) were seeded on top in respective cell culture medium in duplicates of six biological replicates. Viability was analyzed after 1, 3, 7, and 10 days in culture by Invitrogen alamarBlue assay according to the manufacturer's instructions. In brief, the alamarBlue reagent was added to culture medium at a ratio of 1:10, followed by incubation for 3 hours at 37°C and 5% CO₂ (and 5% O₂ for LG-MSCs). Fluorescence of supernatants was measured at 560-nm excitation and 590-nm emission (Spark multimode microplate reader; Tecan Life Sciences, Männedorf, Switzerland).

The capacity of sera obtained from dwarf hamsters after SARS-CoV-2 challenge to neutralize SARS-CoV-2 was assessed in vitro as previously described (107 (link)). After inactivation of complement for 30 min at 56 C°, sera were prepared in duplicates as two fold serial dilutions in Minimum Essential Medium (MEM) supplemented with 10% FBS and penicillin/streptomycin in 96-well cell culture plates (Sarstedt). To each serum dilution and the respective control wells, 40 pfu of SARS-CoV-2 was added and neutralization was allowed to proceed for 30 min at room temperature. Afterwards, approximately 1 × 10⁴ Vero E6 cells were added to each well. Subsequently, the plates were incubated at 37 C° under a 5% CO₂ atmosphere for 3 days, fixed with 4% formaldehyde and stained with 0.75% crystal violet (aqueous solution) for quantification of cytopathic effects (CPE). Virus neutralization was considered successful in wells with no evidence of CPE and the last effective serum dilution was counted.

225 μl lithium heparin blood from different donors were pipetted into 96-well cell culture plates (SARSTEDT, Nümbrecht, Germany) and stimulated for 24 hours with 25 μl pyrogen solution or vehicle. After incubation at 37°C and 5% CO₂, the 96-well plates were centrifuged at 2272 × g for 10 minutes, the supernatants were collected and frozen at −80°C until analysis. PGE₂ concentration was determined using the Cayman Prostaglandin E₂ Express EIA Kit (Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer’s instructions.

HeLa and HEL 299 cells (10⁴/well), cultured in 96-well cell culture plates from Sarstedt (Nümbrecht, Germany) for 24 h, were treated with the 1% FBS DMEM medium containing 20 µM C₆₀ for 24 h and exposure to the 1 MHz US treatment. The control cells were treated without and with an equal volume of sterile water as a solvent of C₆₀ colloid solution. Cell viability was determined with an MTT reduction assay [65 (link)] at 48 h after US treatment. Briefly, cells were incubated for 2 h at 37 °C in the presence of 0.5 mg/mL MTT. The diformazan crystals were dissolved in DMSO and determined at 570 nm with a microplate reader Tecan Infinite M200 Pro (Männedorf, Switzerland).
Cell viability assay was accompanied by the phase contrast microscopy analysis of cells under the study with the Keyence BZ-9000 BIOREVO (Osaka, Japan).