To assess changes in neuronal differentiation, HPC0A07/03C cells were plated into clear 96-well plates (Nunclon) at a density of 1.2x104 cells/well. Twenty-four hours later, cells were cultured in the presence of EGF, bFGF, and 4-OHT and with IFN-α, or other proteins/drugs for 3 days (see supplementary Materials). After this initial proliferation phase, cells were washed and then cultured in media containing IFN-α or the same proteins/compounds but without growth factors or 4-OHT for 7 subsequent days. This paradigm was used for all experiments. Finally, cells were rinsed with warm PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature.
Clear 96 well plates
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Clear 96-well plates are a standard laboratory equipment used for various assays and experiments. They provide a clear, transparent surface for visual observation and analysis of samples. The plates are made with a consistent thickness and quality to ensure reliable and reproducible results.
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Lab products found in correlation
Infinite 200 pro, by Tecan (2 mentions)
Icycler, by Bio-Rad (2 mentions)
Sypro orange, by Thermo Fisher Scientific (2 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Picogreen dsdna reagent and kit, by Thermo Fisher Scientific (1 mentions)
Multiskan go, by Thermo Fisher Scientific (1 mentions)
Papain, by Merck Group (1 mentions)
Shark chondroitin sulfate, by Merck Group (1 mentions)
M200 microplate reader, by Tecan (1 mentions)
Cell discoverer 7 microscope, by Zeiss (1 mentions)
Prism 8.3.0 for windows, by GraphPad (1 mentions)
8 protocols using clear 96 well plates
1
Neuronal Differentiation Assay in HPC0A07/03C Cells
2
Quantifying Cartilage Extracellular Matrix Components
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Tissues were digested through the addition of 0.25 mg papain (Sigma) solution directly to each tube or well followed by overnight incubation at 60°C. The digest was used to quantify both the DNA and the sGAG in tissue constructs. The medium collected during the medium exchanges was analyzed to determine the quantity of secreted sGAG. 1,9 Dimethyl methylene blue zinc chloride double salt (DMB, Sigma) was used for sGAG quantification. Digests or culture medium were dispensed in clear 96-well plates (Nunc), then DMB dye was added and the signal was measured at 590 using a plate reader (MULTISKAN GO, Thermo Fischer). Shark chondroitin sulfate (Sigma) was used to generate a standard curve.
PicoGreen dsDNA Reagent and Kit (Invitrogen) was used to quantify the DNA content in the micropellet digests. The papain digest and the PicoGreen solution was mixed in a black 96-well plate (half-area, Costar) and measured in a plate reader (FLUOstar OMEGA, BMG Labtech) at an excitation and emission of 480 nm and 520 nm, respectively.
PicoGreen dsDNA Reagent and Kit (Invitrogen) was used to quantify the DNA content in the micropellet digests. The papain digest and the PicoGreen solution was mixed in a black 96-well plate (half-area, Costar) and measured in a plate reader (FLUOstar OMEGA, BMG Labtech) at an excitation and emission of 480 nm and 520 nm, respectively.
3
Improved Enzymatic Activity Assay for Recombinant GSTs
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The enzymatic activity assay for recombinant GSTs protein was improved by using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate and referring to the method of Li et al. [20 (link)]. Different concentrations of CDNB (0.05–1.6 mM), 1 μg protein and GSH diluent (1 mM, dissolved in 100 mM phosphate buffer) were pipetted in the clear 96-well plates (ThermoFisher, Waltham, MA, USA). The absorbance was measured every 1 min for 6 times at 340 nm using an M200 Microplate Reader (Tecan, Männedorf, Switzerland).
Inhibition assays were carried out in the same manner as the above enzyme activity assay test. Serial dilutions of (S)-(−)-palasonin were mixed with 1 μL CDNB (1 mM), 1 μg recombinant protein and 100 μL PBS (100 mM) at 25 °C for 10 min prior to the addition of the substrate GSH. The reaction was then initiated by sequentially adding 1 μL of GSH (1 mM) and PBS (100 mM), resulting in a total volume of 200 μL of the reaction system.
The relative inhibition was calculated as follows:
In this formula, NC represents the peak area of the control group and CT represents the peak area of (S)-(−)-palasonin treatment group.
Inhibition assays were carried out in the same manner as the above enzyme activity assay test. Serial dilutions of (S)-(−)-palasonin were mixed with 1 μL CDNB (1 mM), 1 μg recombinant protein and 100 μL PBS (100 mM) at 25 °C for 10 min prior to the addition of the substrate GSH. The reaction was then initiated by sequentially adding 1 μL of GSH (1 mM) and PBS (100 mM), resulting in a total volume of 200 μL of the reaction system.
The relative inhibition was calculated as follows:
In this formula, NC represents the peak area of the control group and CT represents the peak area of (S)-(−)-palasonin treatment group.
4
PFAS Cytotoxicity Evaluation by MTT Assay
5
PFAS Cytotoxicity Evaluation by MTT Assay
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The cytotoxic effect of PFASs was measured by a colorimetric MTT assay as described previously (Tachachartvanich et al., 2020 ). Briefly, MDA-kb2 cells were plated at a density of 3.0 × 104 cells/well in clear 96-well plates (Thermo Scientific, Waltham, MA). After 24 h incubation, cells were treated with PFASs at concentrations ranging from 0.1 to 100 μM in triplicate per treatment. The optical density was assessed at 570 nm with reference wavelength of 650 nm using microplate spectrophotometer (Tecan Infinite® 200PRO, Austria). All cytotoxicity experiments were performed side-by-side with the corresponding cell culture assays and repeated at least three times in a separate independent setup.
6
SARS-CoV-2 Cytopathic Effect Assay
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20.000 Vero E6 hTMPRSS2 cells per well were seeded in 75 µL media in clear 96-well plates (Thermo Scientific, cat. no. 167008) and left to adhere overnight. Compounds (4-fold, 8-point dilution series) were diluted from 200X in DMSO to 4X in media before addition of 25 µL compound solution to the cells (final DMSO = 0.5%). Cells were treated in triplicate. After 1h treatment, all media was removed and 50 µL DMEM (with no additives) containing MOI 0,05 SARS-CoV-2 was added. After 1 hour 50 µL treatment media was added and left for 72 hours. Before harvest, cells were washed thrice with PBS and fixed with 1,5% Formaldehyde for 1 hour. Cells were then stained with Hoechst 33342n in PBS.
The plates were imaged at 8 sites in each well at 10X magnification in a Zeiss Celldiscoverer 7 microscope and exported and images were abberancies were removed. Cells with condensed chromatin were counted in CellProfiler 3.1.9. The count for the 8 sites each well was summed, all wells were subtracted the mean count from uninfected wells (10 wells), normalized to DMSO/media treated cells and fitted to a 4-parameter nonlinear regression in Prism 8.3.0 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.
The plates were imaged at 8 sites in each well at 10X magnification in a Zeiss Celldiscoverer 7 microscope and exported and images were abberancies were removed. Cells with condensed chromatin were counted in CellProfiler 3.1.9. The count for the 8 sites each well was summed, all wells were subtracted the mean count from uninfected wells (10 wells), normalized to DMSO/media treated cells and fitted to a 4-parameter nonlinear regression in Prism 8.3.0 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.
7
FXR-LBD Thermal Shift Assay
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Thermal shift experiments were performed in clear 96-well plates (Invitrogen) using SYPRO Orange (Invitrogen) as dye as described previously38 (link). 10 μL of test compound (final concentrations: 3 : 100 µM; 16 : 100 µM) in assay buffer (10 mM TRIS (pH 8.3), 5 mM DTT, 0.5 mM EDTA, 100 mM NaCl) were mixed with 10 μL of FXR-LBD (final protein concentration 5 µM) in assay buffer and 5 µL of SYPRO Orange (5 × final concentration). Temperature-dependent fluorescence increase reporting protein denaturation was measured in duplicates in an ICycler (Bio-Rad Laboratories, Hercules, CA, USA) from 20 to 90 °C in steps of 0.5 °C per minute at 300 nm excitation and 570 nm emission wavelength. The first derivative of the melting curve was calculated using the Graph Pad Prism 5 software. All curves were determined at least four times in independent experiments. Results (expressed as mean ± SEM; n ≥ 4): 3 : 100 µM: 3.75 ± 0.25 °C; 16 : 100 µM: −1.0 ± 0.0 °C; 3 : 100 µM + 16 : 100 µM: 6.00 ± 0.33 °C.
8
Thermal Shift Assay for Ligand Binding
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Thermal Shift assay was performed in clear 96-well plates (Invitrogen) using SYPRO Orange (Invitrogen Darmstadt, Germany) as dye. 10 μL of test compound (GW4064: final concentration 1 μM–500 μM, 100 μM for competitive testing; NSAIDs: 500 μM final concentration) in assay buffer (10 mM TRIS (pH 8.3), 5 mM DTT, 0.5 mM EDTA, 100 mM NaCl) were mixed with 10 μL of protein (final protein concentration 5 μM) in assay buffer and 5 μL of SYPRO Orange (5 × final concentration) in assay buffer. Temperature-dependent fluorescence increase reporting protein denaturation was measured in duplicates in an ICycler (Bio-Rad) from 20 to 90 °C in steps of 0.2 °C per minute at 300 nm excitation and 570 nm emission wavelength. Each experiment was independently repeated four times. The first derivative of the melting curves was calculated using the Graph Pad Prism 5 software.
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