Pseudovirus neutralization assay was performed based on previous protocols (Zhao et al., 2013 (link)). Briefly, HIV-1 backbone based pseudovirus was produced in 293T cells by co-transfection with plasmid encoding SARS-CoV-2 S protein and plasmid encoding luciferase expressing HIV-1 genome (pNL4-3.luc.RE) using PEI. Pseudovirus-containing supernatants were collected 48 h later and concentrated using Lenti-X concentrator kit (Takara, CA). Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2 pseudovirus with serially diluted antibodies or ACE2-Fc for 1 h at 37°C, followed by addition of the mixture into pre-seeded 293T-ACE2 cells. The mixture was then centrifuged at 1000 × g for 1 hour at room temperature. The medium was replaced 4 hr later. After 24 h, luciferase expression was determined by Bright-Glo kits (Promega, Madison, WI) using BioTek synergy multi-mode reader (Winooski, VT). Cells only and virus only wells were included and used for normalization. The 50% pseudovirus neutralizing antibody titer (IC50) was calculated using Graphpad Prism 7. Experiments were performed in duplicate and the error bars denote ± 1 SD.
Synergy multi mode reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy Multi-Mode Reader is a versatile laboratory instrument designed for various applications. It offers detection modes such as absorbance, fluorescence, and luminescence, enabling researchers to perform a wide range of assays and experiments. The Synergy Multi-Mode Reader provides reliable and accurate data collection to support scientific investigations.
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Lab products found in correlation
Lenti x concentrator kit, by Takara Bio (2 mentions)
Bright glo kit, by Promega (2 mentions)
Prism 7, by GraphPad (2 mentions)
Caspase glo 3 7 kit, by Promega (2 mentions)
Mtt assay, by Thermo Fisher Scientific (2 mentions)
Caspase glo 3 7, by Promega (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Gsh gssg glo assay, by Promega (2 mentions)
Ros glo h2o2 assay, by Promega (2 mentions)
L 8900 amino acid analyzer, by Hitachi (2 mentions)
Gel extraction, by Qiagen (2 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (2 mentions)
Attune nxt acoustic focusing flow cytometer, by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
G lisa rho activation assay biochem kit, by Cytoskeleton (1 mentions)
Prism 5, by GraphPad (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Abts solution, by Merck Group (1 mentions)
Cell death detection elisa kit, by Merck Group (1 mentions)
Fluorobrite medium, by Thermo Fisher Scientific (1 mentions)
42 protocols using synergy multi mode reader
1
SARS-CoV-2 Pseudovirus Neutralization Assay
2
Caspase-3 and Caspase-7 Assay in MCF-7 Cells
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The effect of the most bioactive compounds (2 ), (6 ) and (9 ) were used for the analysis of caspase-3 and caspase-7 activities on MCF-7 cells by using the Caspase-Glo® 3/7 kit (Promega, Germany). In fact, MCF-7 cells were seeded at a density of 104 cells per well on 96-well microtiter plates, and the plates were incubated overnight at 37 °C in a 5% CO2 humidified environment. Then, the cells were exposed to the active compounds at different concentrations (½×IC50, IC50 and 2×IC50) or DMSO (0.5%) used as negative control, and further incubated for 18 h at 37°C in a 5% CO2 humidified environment. Thereafter, 100 µL of Caspase-Glo® 3/7 reagent was added to each well, mixed and incubated in the dark for 1 h at room temperature. The luminescence was then measured on a microplate reader (Synergy Multi-Mode Reader, BioTek, Winooski, United States of America). The caspase-3/-7 activity was expressed as fold change of cells treated with 0.5% DMSO (control).
3
Quantitative analysis of transgene expression
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Transformants were picked from the selective plate and inoculated into liquid QATM medium and grown for 2 days. 100 µL aliquots of both the whole culture and the cell-free supernatant were separately loaded onto Corning black sided clear bottom 96-well microplates. Optical density (OD600), GFP fluorescence (Ex: 485/20 nm, Em: 520/20) and chlorophyll fluorescence (Ex: 360/40 nm, Em: 680/30 nm) of each well was determined using Synergy Multi-Mode Reader (BioTek Instruments). Attune NxT acoustic focusing flow cytometer (ThermoFisher Scientific) was used to measure the relative per cell GFP (530/30 nm) and chlorophyll (695/40 nm) fluorescence. For both microplate assays and flow cytometry, chlorophyll fluorescence was measured simultaneously to control for potential differences caused by cell physiology/autofluorescence.
4
Caspase-mediated Apoptosis Induction in Cancer Cells
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The induction of apoptosis by the most active extracts from each plant was evaluated by measuring the caspase 3/7 activity on different cancer cell lines with the Caspase-Glo® 3/7 assay kit (Promega). All four cancer cell lines were seeded at a density of 104 cells per well on 96-well microtitre plates, and were allowed to adhere overnight. These cells were treated with the extracts at different concentrations (½ × IC50, IC50 and 2 × IC50) or DMSO (0.5%) as negative control, and the plates were incubated at 37 °C with 5% CO2 for 24 h. After treatment, the Caspase-Glo® 3/7 was prepared according to manufacturer’s guidelines, and 100 μL of the reagent was added per well and incubated for 1 h at room temperature in the dark. Following this incubation, the luminescence was measured on a microplate reader (Synergy Multi-Mode Reader, BioTek). The data was analysed, and expressed as percentage of the untreated cells (control) and fold change.
5
Rho GTPase Activity Assay
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The intracellular Rho activity was detected by using a commercial G-LISA Rho activation assay biochem kit (#BK121, Cytoskeleton, USA) according to the manufacturer’s instructions. The activity of Rho was reflected by fluorescence intensity which could detect through a microplate luminescence reader (SYNERGY multi-mode reader, BioTek Instruments, USA).
6
MTT Assay for Cell Viability
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Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Invitrogen), as described in our previous study [38 (link)]. Briefly, cells were seeded in KBM-2 basal media in a 96-well plate overnight in a 37 °C incubator with 5% CO2. The cells were incubated with varying concentrations of PVP-I (0%, 0.01%, 0.05%, 0.1%, 0.5%, 1% and 2%) for 1 min, followed by three washes with 1X
PBS and supplemented with fresh KBM-2 medium. The cells were incubated for 24 h at 37 °C, followed by the addition of 100 μL of MTT reagent (5 mg/mL in PBS) to each well for 4 h at 37 °C. The supernatant was then aspirated, followed by the addition of 100 μL of cell lysis buffer (20% SDS in 50% DMF) for an hour. The absorbance was measured using a microplate reader (Synergy multi-mode reader, BioTek, Winooski, VT, USA) and the cell viability was expressed as a percentage over control and calculated using the formula (mean OD of treated cells/mean OD of untreated control cells) × 100 and expressed as cell viability (%).
PBS and supplemented with fresh KBM-2 medium. The cells were incubated for 24 h at 37 °C, followed by the addition of 100 μL of MTT reagent (5 mg/mL in PBS) to each well for 4 h at 37 °C. The supernatant was then aspirated, followed by the addition of 100 μL of cell lysis buffer (20% SDS in 50% DMF) for an hour. The absorbance was measured using a microplate reader (Synergy multi-mode reader, BioTek, Winooski, VT, USA) and the cell viability was expressed as a percentage over control and calculated using the formula (mean OD of treated cells/mean OD of untreated control cells) × 100 and expressed as cell viability (%).
7
MTT-Based Cell Viability Assay
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Cell viability assay was carried out as mentioned previously [38 (link)]. Briefly, HNSCC cells were seeded into 96 well plates at a density of 4 × 103 cells/well.
Cell lines were left overnight to attach, then treated with decreasing concentrations of paclitaxel in duplicates. Following this, 72 h after treatment, medium was removed and 50 μL PBS containing 1 mg/mL 3-(4,5-dimethylthiaziazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to each well and cells were incubated for 1 h at 37 °C. After the incubation MTT solution was removed and tetrazolium crystals were dissolved in isopropanol containing 10% (V/V) Triton X-100 and 1% (V/V) 0.1 N HCl. Absorbance was measured at 570 nm and 690 nm with a Synergy multimode reader (BioTek, Budapest, Hungary). The 690 nm data was subtracted from the 570 nm for each well. Absolute IC50 values were calculated by non-linear regression using Graph Pad Prism 5 software (GraphPad Software, San Diego, CA, USA). Each experiment was repeated at least three times.
Cell lines were left overnight to attach, then treated with decreasing concentrations of paclitaxel in duplicates. Following this, 72 h after treatment, medium was removed and 50 μL PBS containing 1 mg/mL 3-(4,5-dimethylthiaziazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to each well and cells were incubated for 1 h at 37 °C. After the incubation MTT solution was removed and tetrazolium crystals were dissolved in isopropanol containing 10% (V/V) Triton X-100 and 1% (V/V) 0.1 N HCl. Absorbance was measured at 570 nm and 690 nm with a Synergy multimode reader (BioTek, Budapest, Hungary). The 690 nm data was subtracted from the 570 nm for each well. Absolute IC50 values were calculated by non-linear regression using Graph Pad Prism 5 software (GraphPad Software, San Diego, CA, USA). Each experiment was repeated at least three times.
8
Caspase-3/7 Activation in Virus-Infected Cells
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A172, D54, U138, U87, or D458 cells were seeded at a density of 2 × 103 cells per well on 96-well plates and allowed to adhere overnight. Cells in triplicate wells were infected with 5 PFUs/cell of HSV-1(F), R3616, or C5252 virus, and the plates were incubated at 37°C with 5% CO2 for 8 h. After treatment, the Caspase-Glo 3/7 (Promega, Madison, WI, USA) was prepared according to the manufacturer’s guidelines, and 100 μL of the reagent was added per well and incubated for 1 h at room temperature in the dark. The luminescent signal was recorded by a multimode microplate reader (Synergy Multi-Mode Reader, BioTek).
9
3T3-L1 Cell Viability Assay
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3T3-L1 cells were seeded in 96-well plates at a density of 7.5 ×
102 cells/well containing 200 μL of 10% FBS-DMEM; high
glucose. After cell seeding, P. dentata 50% MeOH extract was
added by concentration dependent (6.25, 12.5, and 25 μg/mL). After 24 and
48hr after addition of the extract, alamarBlueTM Cell Viability
Reagent (ThermoFisher Scientific, Waltham, MA, USA) was added and then
fluorescence value was measured by SYNERGY multi-mode reader (BioTek, Seoul,
Korea). Viability of cells was measured using alamarBlue assay according
manufacturer instructions.
102 cells/well containing 200 μL of 10% FBS-DMEM; high
glucose. After cell seeding, P. dentata 50% MeOH extract was
added by concentration dependent (6.25, 12.5, and 25 μg/mL). After 24 and
48hr after addition of the extract, alamarBlueTM Cell Viability
Reagent (ThermoFisher Scientific, Waltham, MA, USA) was added and then
fluorescence value was measured by SYNERGY multi-mode reader (BioTek, Seoul,
Korea). Viability of cells was measured using alamarBlue assay according
manufacturer instructions.
10
Quantifying NET Release and MPO-DNA Complexes
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