Pseudoviruses (virus-like particles pseudotyped with the SARS-CoV-2 spike protein) were prepared by co-transfection of HEK293 cells with 1 µg pNL4-3.luc.R-E- plasmid (luciferase expressing HIV-1 with defective envelope protein) (NIH AIDS Reagent Program, ARP2128) and 0.06 mg CMV promoter-driven plasmid encoding the spike protein using Lipofectamine 2000 transfection reagent (ThermoFisher, 11668027), exactly as described [23 (link)]. The infection assay was similarly performed as described [23 (link)], in brief, by pre-incubating pseudovirus with serial dilutions of nBio from either cell culture supernatant or mouse plasma in media at RT for 30 min, prior to addition to HEK293T cells stably expressing full-length human ACE2 protein. The cells and nBio/pseudovirus mixture were incubated at 37 °C with 5% CO2 for six hours, after which the media was replaced with fresh DMEM (10% FBS and 1% penicillin–streptomycin). After 72 h, DMEM was removed and DPBS (ThermoFisher) was added to cells before mixing with an equal volume of ONE-Glo EX Luciferase Assay System (Promega E8130), shaking for 5 min at room temperature, then reading the luciferase signal using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data were analyzed by GraphPad Prism Version 8.4.3 (GraphPad Software, LLC) to obtain IC50 and IC90 values.
Biotek synergy neo plate reader
Manufactured by Agilent Technologies
Sourced in United States
The BioTek Synergy Neo is a multi-mode microplate reader that can perform absorbance, fluorescence, and luminescence detection. It offers a wide wavelength range and versatile detection capabilities for various assay types.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
One glo ex luciferase assay system, by Promega (1 mentions)
Prism version 8, by GraphPad (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Quantichrom atpase gtpase assay kit, by BioAssay Systems (1 mentions)
4 protocols using biotek synergy neo plate reader
1
SARS-CoV-2 Spike Pseudovirus Neutralization Assay
2
Fluorescence Polarization Assay for Tpx2-AURKA Binding
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FITC-Tpx21–43 and unlabeled Tpx21–43 were synthesized by Proteogenix (Schiltigheim, France). Unlabeled proteins and FITC-Tpx21–43 were mixed in FP buffer (25 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.1 mg/mL BSA (Sigma), 0.01% Brij, 3 mM ADP (Sigma), 2 mM DTT) in a 384-well flat-bottom black plate (Corning 3573) at a final volume of 25 µL. For Kd extraction, FITC-Tpx21–43 probe was kept at 5 nM throughout the experiments and each analyzed protein was added to the concentrations indicated in each figure. Each data point was derived from a technical duplicate and each binding curve was performed in triplicate. For the competitive displacement assay, FITC-Tpx21–43 (5 nM) and AURKA protein concentrations (35 nM) were kept constant throughout experiments, and unlabeled competitor (either Bora or Tpx2) was added to final concentrations indicated in each figure. Fluorescence polarization was measured on a BioTek Synergy Neo plate reader (BioTek) using Gen5 v2.05 software with excitation and emission at 485/528 nm, respectively. Binding graphs and the derived binding constants were generated using GraphPad Prism v8.1.2 and v8.3 (GraphPad).
3
Hsp90α ATPase Activity Assay
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In brief, ATPase activity assay was performed by mixing Hsp90α solution (5 μL, 6 μM in assay buffer), 20 μL assay buffer, ATP solutions (10 μL, 4 mM in assay buffer), and 5 μL of tested compounds (14 and BIIB021) to yield final concentrations of 50 μM for 30 min at the room temperature using the QuantiChrom ATPase/GTPase Assay Kit (BioAssay Systems, Hayward, CA, USA). The reaction was terminated by adding 200 μL reagent buffer. Followed by incubation with reagent buffer for 30 min at the room temperature, the absorbance was measured at 620 nm using a Biotek-Synergy Neo-Plate Reader (BioTek Instruments, Winooski, VT, USA).
4
Protein-RNA Binding Assay for AtALKBH9B and ECT2
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The AtALKBH9B and ECT2 were amplified by PCR and cloned into the modified pCAMBIA_nLUC and pCAMBIA_cLUC vectors containing the 35S promoter (primers are listed in Supplementary Table 1). The pro35S::9B:nLUC and pro35S::cLUC:ECT2 constructs were transformed into the Agrobacterium tumefaciens strain GV3101 and then infiltrated into Nicotiana benthamiana leaves along with P19.
The detached leaves were sprayed with 1 mM luciferin (GLPBio) at 2 days after infiltration. The luminescence signal was visualized with a Tanon-5200 (Tanon), and the images were acquired and processed using AllCap software (Tanon). assay was carried out following the previously described method (Harrison et al., 2016) .
Binding assays were performed in 25 mM HEPES (pH 7.5) and 100 mM NaCl including 10 nM FAM-labeled RNA oligonucleotide (GGCCAACUACGU and GGCCAm 6 ACUACGU) in black and flat-bottom 96-well plates (BBI). Proteins were serially diluted 2-fold and the final assay volume was 25 µL per well. The signal was detected at room temperature on a BioTek Synergy Neo plate reader (BioTek).
Polarization (P) was converted to anisotropy (A) using the formula A = 2P/(3-P). Data were plotted as fraction bound by setting the highest anisotropy measured to 1. Data were plotted using GraphPad Prism (version 6.0), and dissociation constants (Kd) were obtained by fitting the curve to a non-linear regression model.
The detached leaves were sprayed with 1 mM luciferin (GLPBio) at 2 days after infiltration. The luminescence signal was visualized with a Tanon-5200 (Tanon), and the images were acquired and processed using AllCap software (Tanon). assay was carried out following the previously described method (Harrison et al., 2016) .
Binding assays were performed in 25 mM HEPES (pH 7.5) and 100 mM NaCl including 10 nM FAM-labeled RNA oligonucleotide (GGCCAACUACGU and GGCCAm 6 ACUACGU) in black and flat-bottom 96-well plates (BBI). Proteins were serially diluted 2-fold and the final assay volume was 25 µL per well. The signal was detected at room temperature on a BioTek Synergy Neo plate reader (BioTek).
Polarization (P) was converted to anisotropy (A) using the formula A = 2P/(3-P). Data were plotted as fraction bound by setting the highest anisotropy measured to 1. Data were plotted using GraphPad Prism (version 6.0), and dissociation constants (Kd) were obtained by fitting the curve to a non-linear regression model.
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