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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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)
Sytox green, by Thermo Fisher Scientific (4 mentions)
Gene5, by Agilent Technologies (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Cell counting kit 8 (cck8), by Beyotime (3 mentions)
Dmi3000b, by Leica (3 mentions)
Vialight plus kit, by Lonza (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)
500 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
Schwann Cell Viability Assay
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Schwann cells were seeded onto 96-well culture plates at a density of 2 × 105 cells/mL and exposed to Cell Counting Kit-8 solution using Cell Counting Kit-8 (CCK-8; Beyotime). At 2 hours after CCK-8 exposure, absorbance at 450 nm was measured using a SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Burlington, VT, USA).
5
Cell Death Assay Protocol
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Cell death assays were described previously [52 (link)]. Briefly, 2000 cells were seeded in each well of the 96-well plate with Dox if needed. On the second day, cells were treated with different necroptotic inducers in triplicate with or without inhibitorsfor indicated times. CellTiter-Glo assays were carried out according to the manufacturer’s instructions (Promega) and luminescent data were recorded with SynergyTM 2 Multi-Mode Microplate Reader from BioTek. The percentage of cell survival was calculated as (luminescence signal in inducer-treated wells/luminescence signal in DMSO-treated wells) *100. For imaging, 1 μM of Sytox Green (S7020, ThermoFisher) and 100 μg/ml Hoechst (H3570, ThermoFisher) were applied. The images were taken with a Cytation 3 imaging reader from BioTek. Except otherwise specified, 20 ng/ml TNFα, 100 nM Smac-mimetic and 20 μM Z-VAD-FMK were used to induce cell death in HT-29 cells. For NTD-DmrB cells, 20 nM dimerizer and 20 μM Z-VAD-FMK were used.
6
Fluorescent Biomolecule Immobilization on Titanium
7
Cell Viability and Cytotoxicity Assays
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Cells were seeded onto 96-well plates at a density of 30,000 cells per well. The second day after seeding, the cells were counted and pretreated with 20 μM Z-VAD-FMK (Beyotime Biotechnology), 1 μM necrosulfonamide (MCE, Monmouth Junction, NJ, USA), 1 μM GSK’872 (MCE), 10 μM necrostatin-1 (MCE), or DMSO vehicle control for infection or transfection. LDH released was determined by the LDH cytotoxicity assay detection kit (Beyotime Biotechnology). The cell culture supernatant was collected for detection. The absorbance was read at 490 nm with Infinite® F50 Absorbance Microplate Reader (Tecan, Switzerland). Cell viability was measured based on the intracellular ATP levels using CellTiter-Lumi™ Plus Luminescent Cell Viability Assay Kit (Beyotime Biotechnology). An equal volume of Celltiter-LUMI™ Plus reagent was added to the cell culture medium to induce cell lysis by oscillation. After incubation at room temperature, the luminescence signal was measured by SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).
8
Bioluminescent ATP Quantification for Cell Viability
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Bioluminescent detection of cellular adenosine triphosphate (ATP) as a measure of cell viability was undertaken using ViaLight® Plus Kit (Lonza Inc.) reagents. Cells were plated at optimal seeding density to ensure exponential growth (4 × 103 cells per well) in 96-well, white-walled plates. Wells were dosed with agents to a final volume of 100 µL. Dosing occurred at the beginning of the experiment, and cells were incubated for up to 120 h. Luminescence was detected with SynergyTM 2 multi-mode microplate reader (BioTek® Instruments). Each experiment was performed in at least triplicate wells in triplicate experiments.
9
Evaluating Schwann Cell Viability
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Schwann cells transfected with CD146 siRNA or control siRNA for 36 h were seeded on 96-well plates at a density of 2 × 105 cells/ml and exposed to 10 μl of CCK8 using Cell Counting Kit-8 (Beyotime, Shanghai, China). After 2 h culture, cell viability was measured spectrophotometrically at 450 nm using a SynergyTM 2 Multi-Mode Microplate Reader (BioTek, Burlington, VT, United States). Experiment was conducted four times using independent cultures, each tested induplicate.
10
Complement-mediated Cell Viability Assay
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Cells at 5,000 cells/well were seeded in 96-well plates overnight. Following treatment with human serum complement with and without the presence of chemical inhibitors, cells were incubated in 0.5 mg/ml of MTT solution (Sigma) at 37°C for 4 h. The solution was then replaced with DMSO. The optical densities were determined at 540 nm and 630 nm using a Synergy TM 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT).
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