Multiskan ex plate reader

Test sera were diluted with the 1% BSA-PBS, applied as serial dilutions in the 1:20–1:160 range to the RBD-coated plates (RBD:ACE2-HRP test design) and incubated in plate wells for 30 min at 37 °C, if not stated otherwise in the Results section. ACE2-HRP conjugate was used at 0.7 nM concentration (corresponds to 62.5 ng/mL of the ACE2) in the case of RBD antigen and 2.9 nM (250 ng/mL) in the case of beta-RBD antigen, if not stated otherwise in the Results section. In the case of the ACE2:RBD-HRP test design, test serum samples were diluted as 1:20–1:160, pre-incubated with RBD-HRP in polypropylene test tubes (RBD-HRP concentration 14.2 nM, corresponds to 390 ng/mL of the RBD) for 30 min, transferred to ACE2-coated ELISA plates and incubated further for 30 min.
Wells were washed five times with the PBST. The color was developed for exactly 10 min at room temperature (+25 ± 2 °C), utilizing the ready-to-use TMB solution (Xema Co., Ltd., Moscow, Russia), 100 μL/well. The reaction was stopped with 100 μL of 5% orthophosphoric acid. Optical density at 450 nM was measured using the Multiskan EX plate reader (Thermo Fischer Scientific, Waltham, MA, USA).

After 48 h of treatment with either BB-CLA (0 to 20 μM) or DMSO (control), a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro toxicology assay (Sigma-Aldrich) was carried out per manufacturer’s instructions and absorbance was measured at 570 nm on a Multiskan EX plate reader (Thermo Fisher Scientific). Optical densities of wells treated with BB-CLA were compared with those treated with equivalent amounts of DMSO to determine cell viability. Values were expressed relative to DMSO treated wells.

The cells were seeded in a 96-well plate (at a density of 2 × 10³ cells for HA22T/VGH; 1.5 × 10³ cells for HuH7) and exposed to various concentrations of didox and only HA22T/VGH also to hydroxyurea, DFO or DFP (0, 1, 10, 25, 50, 100, 200 and 500 µM) for 24, 48 and 72 h. In other experiments, HA22T/VGH were seeded in 96-well plates and treated with a single dose of didox, HU, DFO, DFP alone or in combination with increasing doses of FAC (25, 50, 100, 200 and 400 µM) for 48–72 h. In other type of treatment, HA22T/VGH cells were or pre-treated for 16 h with a single dose of didox (200 µM) and then treated in combination with FAC (400–800 µM) or directly in combination didox-FAC for 48–72 h.
Cell viability was evaluated with an MTT assay (Sigma-Aldrich, Saint Louis, MO). After the indicated time points and treatments, the supernatant was removed and 100 µL of the MTT solution (0.5 mg/mL) diluted in the cell medium was added to the wells. After 3.5 h of incubation at 37 °C and 5% CO₂, the MTT medium was removed and 75 µL of DMSO was added to each well. Plates were shaken for 15 min at 37 °C until complete dissolution and absorbance was measured at 540 nm emission wavelengths using a Multiskan^©EX plate reader (Thermo Scientific, Waltham, MA, USA). Average percentage of cell viability at each concentration was calculated using Microsoft Excel 2016 software.

After 48 h of culturing human mammary cell lines in CM, cell line medium, or DMEM, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro toxicology assay or a lactate dehydrogenase (LDH) assay was carried out, as previously described^{11 (link)}, according to manufacturer’s instructions (Sigma). MTT and LDH absorbances were measured at 570 nm and 490 nm, respectively, on a Multiskan EX plate reader (Thermo Scientific, Vantaa, Finland) and background measurements of 690 nm were subtracted for both. Optical densities of wells treated with EqMDEC CM were compared to those treated with either DMEM or control (self-CM) in order to determine cell viability or relative LDH release. Values were expressed relative to wells treated with DMEM and to lysed wells for MTT and LDH release, respectively. For active caspase 3 immunostaining, 4% paraformaldehyde-fixed cells were washed with PBS and treated with 0.5% Triton X-100 for 10 min. Following a 30 min incubation in blocking solution (1% goat serum and 1% BSA in PBS) at RT, fixed cultures were probed with an anti-active caspase 3 primary antibody diluted 1:100 in PBS (ab4051, Abcam, Cambridge, MA) for 1 h, followed by incubation with HRP-conjugated goat anti-rabbit diluted 1:100 in PBS (Jackson ImmunoResearch, West Grove, PA) for 1 h.

Ethyl acetate and H₂O₂ were purchased from BDH, UK. dimethyl sulfoxide (DMSO), 1`1` diphenyl 2 picrylhydrazyl (DPPH), ferric chloride, ferrous sulphate, methanol, 2,4,6-triphyridyl-s-triazine (TPTZ), and 2,2-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid (ABTS), potassium persulfate, ascorbic acid, gallic acid, trolox and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-etrazoliumbromide (MTT) were purchased from Sigma-Aldrich Inc. (USA). Dulbecco’s modified eagle’s medium (DMEM), Rosswell Park Memorial Institute (RPMI) 1640 medium, foetal bovine serum (FBS), penicillin/streptomycin, glutamine, phytohaemagglutinin and cytochalasin B were purchased from Gibco BRL (USA). Bleomycin (BLM) was purchased from United Biotech, India. Brain heart infusion (BHI) broth, BHI agar, blood agar and anaerobic sachets were from Oxoid (UK). Defibrinated sheep blood was from the Veterinary Research Institute, Gannoruwa, Sri Lanka. UV-1800 UV-Vis spectrophotometer, Shimadzu, Japan and Multiskan Ex plate reader from Thermoscientific, USA, were used for antioxidant and MTT assays respectively.

For automated live cell proliferation experiments, the cells were seeded onto 96-well plate: 30 000 VCaP cells and 5 000 22Rv1 cells per well (±ENZ 3w). On the day 0 (d0) of monitoring, the nuclei were stained with IncuCyte NucLight Rapid Red reagent (Essen BioSciences, #4717). The cells were monitored every 8 h in total of 6 days using IncuCyte S3 Live-Cell Imaging System (Essen BioSciences). IncuCyte S3 2021C software (Essen BioSciences) was used to count the number of cells. Results were normalized to d0 and are shown as relative percentage in cell count, representing the mean ± SD of 8 images. For MTS assay, the cells were seeded onto 96-well plates: 30 000 VCaP cells and 10 000 22Rv1 cells per well in charcoal-stripped medium (±ENZ). For RNA interference, the cells were transfected with siNON or siFOXA1 as described above. Wells containing only medium were used to subtract background. The start of the treatments was designated as d0. The cell proliferation was assessed at d0, d1, d2, d3 and d4 using colorimetric CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) kit (Promega, #G3580) according to manufacturer's protocol. The absorbance was measured at 492 nm wavelength using Multiskan EX plate reader (Thermo Scientific). Results were normalized to d0 and are shown as fold change. P-values were calculated using two-way ANOVA with Bonferroni post hoc test.