Caspase-like activity was measured with luminescent assays based on DEVD short peptides with Caspase-Glo 3/7 Assay Kit (Promega). Both caspase 3 and caspase 7 recognize the same DEVD substrate. Briefly, seedlings were harvested after various treatments and total proteins were extracted with liquid nitrogen in a buffer containing 100 mM sodium acetate, pH 5.5, 100 mM NaCl, 1 mM EDTA, and 5 mM DTT. To measure caspase-3/7 activity, 30 µl caspase-3/7 luminogenic substrate (Z-DEVD-aminoluciferin) was added to 50 µg protein extracts and incubated at 22°C for 1 hr protected from light. The luminescence of each sample was measured with the Synergy 2 Multi-Mode Microplate Reader (BioTek). For dual-luciferase activity assays [89] (link), Arabidopsis leaf protoplasts were isolated from 4-week-old soil-grown seedlings and transfected according to a standard protocol [43] (link) with various reporter constructs or cotranstransfected with different effectors. Firefly and renilla luciferase were quantified with Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer's instructions in the Synergy 2 Multi-Mode Microplate Reader (BioTek).
Synergy 2 multi mode microplate reader
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The Synergy 2 Multi-Mode Microplate Reader is a versatile instrument designed for high-performance microplate-based detection and analysis. It is capable of measuring absorbance, fluorescence, and luminescence in a variety of microplate formats.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (9 mentions)
Dual luciferase reporter assay system, by Promega (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Caspase glo 3 7 assay kit, by Promega (5 mentions)
Celltiter glo, by Promega (5 mentions)
Gene5, by Agilent Technologies (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Prism software, by GraphPad (3 mentions)
Munana, by Merck Group (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Griess reagent, by Merck Group (3 mentions)
D6883, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin g, by Thermo Fisher Scientific (3 mentions)
Effectene reagent, by Qiagen (3 mentions)
Costar 96 well assay plate black plate with clear bottom, by Corning (3 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Celltiter 96 aqueous one solution, by Promega (3 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (3 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (3 mentions)
482 protocols using synergy 2 multi mode microplate reader
1
Measuring Caspase-like Activity in Plants
2
Quantifying Nitric Oxide and Cytotoxicity Assays
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Griess Reagent Kit (Thermo Fisher Scienti c) was used to assess NO according to the manufacturer's instructions. Cells were seeded in 96-well plates. We mixed the following reagents in each well: 20 µl Griess Reagent, 150 µl nitrite-containing sample, and 130 µl deionized water. A photometric reference was prepared by mixing 20 µl Griess Reagent and 280 µl deionized water. Absorbance of the nitrite-containing samples relative to the reference was measured in Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, USA) at 548 nm.
For LDH assay, Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scienti c) was used. 50 µl cell culture medium was transferred to a new 96-well plate, 50 µl reaction mixture was added to each well and incubated at room temperature for 30 min, then 50 µl stop solution was added. Absorbance at 490 and 680 nm was measured using Synergy 2 Multi-Mode Microplate Reader (Bio-Tek , USA).
For MTS assay, MTS Cell Proliferation Colorimetric Assay Kit (BioVision, USA) was used. Cells were seeded in 96-well plates and treated as indicated above. After the treatment, cell culture medium of each well was removed and 110 µl reagent containing 10 µl MTS and 100 µl medium was added to each well and incubated in humidi ed air containing 5% CO2 at 37°C for 1 h. Absorbance at 490 nm was measured using a microplate reader.
For LDH assay, Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scienti c) was used. 50 µl cell culture medium was transferred to a new 96-well plate, 50 µl reaction mixture was added to each well and incubated at room temperature for 30 min, then 50 µl stop solution was added. Absorbance at 490 and 680 nm was measured using Synergy 2 Multi-Mode Microplate Reader (Bio-Tek , USA).
For MTS assay, MTS Cell Proliferation Colorimetric Assay Kit (BioVision, USA) was used. Cells were seeded in 96-well plates and treated as indicated above. After the treatment, cell culture medium of each well was removed and 110 µl reagent containing 10 µl MTS and 100 µl medium was added to each well and incubated in humidi ed air containing 5% CO2 at 37°C for 1 h. Absorbance at 490 nm was measured using a microplate reader.
3
Quantifying Nitric Oxide and Cytotoxicity Assays
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Griess Reagent Kit (Thermo Fisher Scienti c) was used to assess NO according to the manufacturer's instructions. Cells were seeded in 96-well plates. We mixed the following reagents in each well: 20 µl Griess Reagent, 150 µl nitrite-containing sample, and 130 µl deionized water. A photometric reference was prepared by mixing 20 µl Griess Reagent and 280 µl deionized water. Absorbance of the nitrite-containing samples relative to the reference was measured in Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, USA) at 548 nm.
For LDH assay, Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scienti c) was used. 50 µl cell culture medium was transferred to a new 96-well plate, 50 µl reaction mixture was added to each well and incubated at room temperature for 30 min, then 50 µl stop solution was added. Absorbance at 490 and 680 nm was measured using Synergy 2 Multi-Mode Microplate Reader (Bio-Tek , USA).
For MTS assay, MTS Cell Proliferation Colorimetric Assay Kit (BioVision, USA) was used. Cells were seeded in 96-well plates and treated as indicated above. After the treatment, cell culture medium of each well was removed and 110 µl reagent containing 10 µl MTS and 100 µl medium was added to each well and incubated in humidi ed air containing 5% CO2 at 37°C for 1 h. Absorbance at 490 nm was measured using a microplate reader.
For LDH assay, Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scienti c) was used. 50 µl cell culture medium was transferred to a new 96-well plate, 50 µl reaction mixture was added to each well and incubated at room temperature for 30 min, then 50 µl stop solution was added. Absorbance at 490 and 680 nm was measured using Synergy 2 Multi-Mode Microplate Reader (Bio-Tek , USA).
For MTS assay, MTS Cell Proliferation Colorimetric Assay Kit (BioVision, USA) was used. Cells were seeded in 96-well plates and treated as indicated above. After the treatment, cell culture medium of each well was removed and 110 µl reagent containing 10 µl MTS and 100 µl medium was added to each well and incubated in humidi ed air containing 5% CO2 at 37°C for 1 h. Absorbance at 490 nm was measured using a microplate reader.
4
Quantifying Beta-Galactosidase Activity
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To determine β-galactosidase activity, cells were grown in the presence or absence of arabinose overnight or for 4–8 h at 30°C. Cells were permeabilized and β-galactosidase activity was measured by the original (36 ) or by a simplified version (37 (link)) of the Miller protocol. In cells grown in microplate wells β-galactosidase activity was determined in Synergy™ HT or Synergy 2 Multi-Mode Microplate Readers (Bio-Tek). Details are described in Supplementary Data. Colonies producing β-galactosidase were identified on X-gal containing agar plates (28 ,36 ).
5
Evaluating Drug Potency in Genetically Engineered Cells
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The half maximal inhibitory concentrations of the selected drugs of HAP1, HAP1 RNF43 KO, and HAP1 PWWP2B KO cells were measured using the MTS assay for selected drugs at concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 µM for 48 h. On the day of the proliferation assay, medium was removed, and 100 μL of fresh medium was added to each well of 96-well plates, followed by 20 μL of MTS solution, and the plates were incubated at 37 °C for 1 h in a humidified environment with 5% CO2. The absorbance was read at 490 nm using a microplate reader (Synergy 2 Multi-Mode Microplate Readers; BioTek, Winooski, VT, USA). The IC50 values were determined after fitting growth inhibition curves to dose-response curves using GraphPad Prism software (GraphPad Software Inc., CA, USA).
6
Evaluating Drug Sensitivity in Gastric Cancer Cells
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The half maximal inhibitory concentrations of the selected drugs of HAP1, HAP1 RNF43 KO and HAP1 PWWP2B KO cells were measured using the MTS assay for selected drugs at concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 µM for 48 h. On the day of the proliferation assay, medium was removed, and 100 µL of fresh medium was added to each well of 96-well plates, followed by 20 µL of MTS solution, and the plates were incubated at 37°C for 1 h in a humidi ed environment with 5% CO 2 .
The absorbance was read at 490 nm using a microplate reader (Synergy 2 Multi-Mode Microplate Readers; BioTek, Winooski, VT, USA). The IC 50 values were determined after tting growth inhibition curves to dose-response curves using GraphPad Prism software (GraphPad Software Inc., CA, USA).
Apoptosis analysis HAP1, HAP1 RNF43 KO, HAP1 PWWP2B KO, SNU620, Kato III, MKN28, and MKN45 cells seeded onto 6well plates at a density 5 × 10 4 cells per mL were treated with the respective IC50 values of docetaxel trihydrate, pelitinib, and uprosertib (Table 2). Cell death was determined using the Annexin V-APC/Propidium Iodide (PI) Apoptosis Detection Kit (Thermo Fisher Scienti c, Rockford, IL, USA) on a CytoFLEX ow cytometer (Beckman Coulter, Brea, CA, USA). The percentages of intact and apoptotic cells were calculated using CytExpert software (Beckman Coulter).
The absorbance was read at 490 nm using a microplate reader (Synergy 2 Multi-Mode Microplate Readers; BioTek, Winooski, VT, USA). The IC 50 values were determined after tting growth inhibition curves to dose-response curves using GraphPad Prism software (GraphPad Software Inc., CA, USA).
Apoptosis analysis HAP1, HAP1 RNF43 KO, HAP1 PWWP2B KO, SNU620, Kato III, MKN28, and MKN45 cells seeded onto 6well plates at a density 5 × 10 4 cells per mL were treated with the respective IC50 values of docetaxel trihydrate, pelitinib, and uprosertib (Table 2). Cell death was determined using the Annexin V-APC/Propidium Iodide (PI) Apoptosis Detection Kit (Thermo Fisher Scienti c, Rockford, IL, USA) on a CytoFLEX ow cytometer (Beckman Coulter, Brea, CA, USA). The percentages of intact and apoptotic cells were calculated using CytExpert software (Beckman Coulter).
7
Bone Marrow Cytokine Profiling
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In this study, we measured the bone marrow concentrations of selected proinflammatory cytokines (IL-1b, IL-2, IL-4, IL-6, IL-17, tumor necrosis factor [TNF]-a, interferon [IFN]-g) and immunosuppressive factors (IL-10, IL-11, and latency-associated peptide 1 [LAP-1]). LAP-1 is part of a latent form of immunosuppressive transforming growth factor (TGF)-b. All cytokine concentrations were measured using ELISA kits (eBioscience, San Diego, CA) according to the manufacturer's manual (to increase assay sensitivity, samples were incubated with capture antibody overnight). Synergy 2 Multi-Mode Microplate Readers (BioTek, Winooski, VT) were used for analysis.
8
Tepotinib IC50 Determination in MKN45 and SNU620
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The IC50 of tepotinib in MKN45 and SNU620 cells was measured using the MTS assay for tepotinib at concentrations of 10, 1, 0.1, 0.05, 0.0025, 0.00125, 0.001, 0.0001, 0.00001, or 0.000001 µM for 48 h. On the day of the proliferation assay, the medium was removed, and 200 µL of fresh medium was added to each well of the 96-well plate, followed by 20 µL of MTS solution, and the plates were incubated at 37 °C for 1 h in a humidified environment with 5% CO2. The absorbance was measured at 490 nm using a microplate reader (Synergy 2 Multi-Mode Microplate Reader; BioTek, Winooski, VT, USA). The IC50 was calculated by nonlinear regression analysis using Prism software (GraphPad Software, San Diego, CA, USA).
9
SYBR Gold Assay for siRNA Polyplex Characterization
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SYBR Gold is a fluorescent dye which is used to stain nucleic acids. It intercalates into siRNA and produces fluorescence. When siRNA is condensed by polymer and becomes inaccessible for SYBR Gold, the fluorescence will decrease. Polyplexes were prepared with bPEI or HAI-SPDP-bPEI and 50 pmol of siRNA at different N/P ratios (1, 2, 3, 5, 7, 10) in 100 µL per well in a FluoroNunc 96 well white plate (Thermo Fisher). Subsequently, 30 µL of 4 × SYBR Gold was added to each well and incubated for 10 min in the dark. The fluorescence of SYBR Gold was determined by a Synergy 2 multi-mode microplate reader (BioTek Instrument, Winooski, VT, USA) with an excitation filter 485/20 nm and an emission filter 520/20 nm. The fluorescence of free siRNA (N/P = 0) represented 100% free siRNA. The experiment was performed in triplicate.
10
Dual-Luciferase Assay for Influenza Protein Expression
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Subconfluent monolayers of 293T cells (7.5 × 105 cells in 35-mm dishes) were transfected with the luciferase reporter plasmid (enhanced green fluorescent protein [EGFP] open reading frame in pHW72-EGFP replaced with the firefly luciferase gene) [45 (link)] and a mix of PB2, PB1, PA and nucleoprotein (NP) expression plasmids (CA/04 or mutated) in quantities of 1, 1, 1, and 2 μg, respectively. The plasmid pGL4.75(hRluc/CMV), which expresses Renilla luciferase (Promega, Madison, WI, USA), was used as an internal control for a dual-luciferase assay. As a negative control, 293T cells were transfected with the same plasmids, with the exception of the NP expression plasmid. After 24 h of incubation at 37°C cell extracts were harvested and lysed, and luciferase levels were assayed with a dual-luciferase assay system (Promega) and a Synergy 2 multimode microplate reader (BioTek Instruments, Winooski, VT, USA). Experiments were performed in triplicate.
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