Quest graph ic50 calculator

Cell cytotoxic assay was performed for testing the cytotoxicity of the RGE against UC-MSCs. Initially, UC-MSCs were re-suspended with the fresh medium and counted. After that, UC-MSCs were seeded on a 96-well plate at the density of 10,000 cells/well and incubated in a CO₂ incubator with 5% CO₂ at 37 °C for 24 h for growth.
The viability rate of UC-MSCs was evaluated through Trypan blue assay like afore-research [20 (link)]. After the above incubation, the culture medium was removed, and these cells were exposed to serial 2-fold dilution of RGE (50 ppm, 100 ppm, 200 ppm, 400 ppm, 800 ppm, 1600 ppm, 3200 ppm, 6400 ppm) for 24 h. After treatment, the treated and untreated cells were stained with a previous procedure [21 (link)]. Briefly, these cells were incubated with 0.4% trypan blue for 10 min at room temperature (RT) after three washing times. These cells were fixed with 4% PFA solution and incubated at RT. Subsequently, the rate of viability cells was obtained by manually counting from the microscopic photos captured.
Finally, the online software named Quest GraphTM IC50 Calculator (AAT Bioquest, Inc., Sunnyvale, CA, USA) [22 ], was used for calculating the IC50 values for UC-MSCs after treatment, such as other research [20 (link),23 (link)].

The cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured according to the ATCC. The human colon cancer cell lines Caco-2 (p53-null) and HT-29 (mutant p53), and non-tumorigenic colon cells CCD18-Co (wild-type p53) were grown as reported elsewhere [47 (link)]. All tested compounds were solubilized in DMSO (<0.5% in the culture medium) and filter-sterilized (0.2 μm) before addition to the culture media. The treatments consisted of a range of 8 concentrations from 0.78 to 100 μM to calculate the concentration that inhibited cell growth by 50% vs. control cells (0.5% DMSO), i.e., IC₅₀ values, on the three cell lines at two incubation times (48 and 72 h). The effects of the compounds on Caco-2, HT-29, and CCD18-Co cell viability and proliferation were measured using the MTT reduction assay according to Giménez-Bastida et al. [47 (link)]. Data are presented as the mean ± standard deviation (SD) of at least three independent experiments (n = 2 wells for each compound (dose and time point) per experiment). Then, IC₅₀ antiproliferative values were determined using a four-parameter logistic regression fit. The calculations were performed using the online tool Quest Graph^TM IC₅₀ Calculator (AAT Bioquest, Inc., Sunnyvale, CA, USA (https://www.aatbio.com/tools/ic50-calculator, accessed on 26 April 2022).

Vero E6 cells were infected with ten-fold dilutions of SARS-CoV-2. Before nebulization, the virus was allowed to attach to cells for 30 minutes at 4 ${}^{\circ }$ C to avoid internalization. Another set of cells was incubated with the same dilutions and allowed to progress for 5 hours at 37 ${}^{\circ }$ C. Ultrasonic nebulization of media was carried out in the previously described sealed chamber for 10 min at room temperature in a safety class-II hood. Upon treatment, the cells were washed and covered with semisolid CMC medium as for plaque assay. Viral plaques were visualized after 72 h and 296 counted as described. In Vero E6 cells measurements of cell viability were performed using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay System (Promega). PAM dilutions were made in distilled water (for PAW) or in PBS (for bPAW). SARS-CoV-2/PAM mixtures were incubated for 30 min at room temperature. Upon incubation, the mixtures were 10-fold serially diluted in DMEM 10% FBS and added to Vero E6 cell monolayers for plaque assay titration as described previously. For ${\text{IC}}_{50}$ estimation we used a four parameter logistic regression model. The calculations were performed using an online tool “Quest Graph ${}^{\mathrm{TM}}$
${\text{IC}}_{50}$ Calculator, AAT Bioquest, Inc (https://www.aatbio.com/tools/ic50-calculator). Values were normalized dividing by the largest response.