Biotek cytation 5 imaging reader

All binding assays were conducted in light gray, half area 96-well plates (PerkinElmer, 6002350) in a total volume of 40 μl. All stock solutions were prepared in the assay buffer comprised of 1× AlphaLISA Epigenetics buffer (PerkinElmer, 5× AL008C/F) with 0.05% v/v Tween 20 and 2 μM tris(2-carboxyethyl)phosphine. For initial screens, each recombinant His₆-tagged PBRM1-BD4 mutant was incubated at three concentrations (0.1 μM, 1 μM, and 10 μM) with 50 nM biotinylated histone H3K14ac(1–20) peptide (NH₂-ARTKQTARKSTGGK(acetyl)APRKQLK(biotin)-CONH₂; Peptide 2.0). Ten microliters of a 4 × (200 nM) biotinylated histone H3K14ac peptide stock solution and 10 μl of 4 × (40, 4, 0.4 μM or 0.004–8 μM) protein stock solutions were added to each well. The plate was then incubated for 30 min at room temperature. A bead solution comprising 8 μg/ml streptavidin donor beads and 8 μg/ml nickel-chelate acceptor beads (PerkinElmer, AlphaScreen Histidine (Nickel Chelate) Detection Kit, 500 assay points, 6760619C) was prepared in assay buffer. Twenty microliters of bead solution was added to each well under reduced light, and the plate was covered and incubated for an additional hour in the dark. Luminescence was subsequently read on a BioTek Cytation 5 imaging reader (Agilent, 16277) using the Alpha filter cube (Agilent, 1325000), and Alpha counts were analyzed using GraphPad Prism.

Neutralizing antibody titers against SARS‐CoV‐2 in patient serum samples were determined using a pseudovirus assay as previously described.³ Briefly, SARS‐CoV‐2 pseudoviruses were generated by co‐transfection of a lentiviral packaging plasmid, lentiviral reporter plasmid expressing green fluorescent protein and luciferase, and pcDNA3.1 expression vectors encoding the S proteins of SARS‐CoV‐2. Viral supernatants were collected 48 hours post‐transfection. Neutralization assays were performed by combining pseudoviruses and serum (at a starting dilution of 1:40) and incubation at 37°C for one hour. The serum/virus mixture was then added to 293/hACE2 cells in triplicate wells followed by centrifuging the cells at room temperature at 800 rpm for one hour and then incubation for 48 hours at 37°C (5% CO₂). Luminescence was measured using Steady‐Glo Luciferase Assay System (Promega) and a BioTek Cytation 5 imaging reader (BioTek).

Lactate dehydrogenase (LDH) and 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed. For the LDH assay, the cells were grown in a 96-well plate (10,000 cells/well); then, they were exposed to CHL (15 µM), BER (10 μM), MAT (10 μM), or TAB (10 μM) for 24 h. The plate was centrifugated (4 mn, 2000 rpm); 50 μL of supernatant was put in a new plate, and 50 μL of reagent from the commercial kit (CytoTox 96^®Non-Radiaoactive Cytotoxicity Assay, G1782, Promega, Madison, WI, USA, G1782) was added. The reaction was stopped 30 mn later, and the optical density was measured at 490 nm (BIOTEK Cytation 5 imaging reader, Winooski, VT, USA). For the MTT assay, the cells were grown in a 96-well culture plate (10,000 cells/well) and were exposed to CHL (15 μM), BER (10 μM), MAT (10 μM), or TAB (10 μM) for 20 h. Twenty microliters of sterile filtered MTT solution (5 mg/mL) was added to each well. After 4 h, the plate was centrifugated (4 mn, 2000 rpm), the medium was removed, and 200 µL of dimethyl sulfoxide was added to resuspend the formazan crystals. The optical density was measured at 560 nm with a reference at 670 nm (BIOTEK Cytation 5 imaging reader, Winooski, VT, USA).

To investigate cytotoxicity, cells were treated for 24 h with either IFN-γ (20 ng/mL), TGF-β1 (20 ng/mL), LPS (1 µg/mL), CHO C3a F3 clone or control CHO supernatants (2% and 20%). Lactate dehydrogenase (LDH) levels released by lysed cells in the culture medium were measured using a colorimetric-based kit (ref. G1781, CytoTox 96^® Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI, USA). We evaluated cytotoxicity as the ratio of released LDH levels after treatments compared to the maximum LDH release induced by Triton 1% exposure.
We also evaluated cell mitochondrial metabolic activity, performing an MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, according to the method of Mosmann [33 (link)]. Cells were cultured (4 × 10³ cells/well) in 96-well plates for 24 h before medium removal and treatment with either IFN-γ (20 ng/mL), TGF-β1 (20 ng/mL), LPS (1 µg/mL), CHO C3a F3 clone or control CHO supernatants (2% and 20%) for 19 h. The culture medium was then supplemented with 20 µL of sterile filtered MTT solution (5 mg/mL in phosphate-buffered saline (PBS)) (Sigma-Aldrich, Darmstadt, Germany) for 5 h. Then, the culture medium was removed and formazan crystals were solubilized in 200 µL of dimethyl sulfoxide. Finally, the absorbance was read at 560 nm (BIOTEK Cytation 5 imaging reader).