Lactate dehydrogenase (LDH) release is an indicator of cellular necrosis. Culture medium was collected for analysis of LDH release using a colorimetric assay kit (Jiancheng, Nanjing, China) following the manufacturer’s protocol. Released LDH was measured by a coupled enzymatic reaction, which results in the conversion of the tetrazolium salt to red formazan by diaphorase. Briefly, 120 μL of cell culture medium was mixed with 60 μL of LDH working solution and incubated for 60 min at room temperature in dark. Absorbance was measured at 490 nm by Thermo Microplate Reader (Thermo, USA). The percentage of LDH release was calculated according to the formula provided by the kit supplier.
Thermo microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo microplate reader is a versatile laboratory instrument designed to measure the absorbance, fluorescence, and luminescence of samples in microplates. It is capable of performing a wide range of assays, including enzyme-linked immunosorbent assays (ELISA), cell-based assays, and protein quantification. The microplate reader provides accurate and reliable data to support various research and analytical applications.
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10 protocols using thermo microplate reader
1
Quantifying Cell Necrosis via LDH Release
2
Evaluating Cell Viability with CCK8 Assay
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A CCK8 assay was used to evaluate the viability of the cells. HCT116 and LS174T cells were seeded into a 96-well plate. TKT inhibitor N3PT treatment (DMSO group, 5 μM N3PT group, 10 μM N3PT group) and siRNA transfection were performed. After 24 h of treatment with N3PT or 72 instructions (Dojindo, Kumamoto, Japan). The optical density of the cells was measured by a Thermo microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.
3
Quantification of Cytokine Levels
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The contents of IL-1β, IL-6, and IL-12 in the cellular medium were determined using commercial ELISA kits purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd., (Shanghai, China). A Thermo microplate reader (Thermo Fisher Scientific Inc., Rockford, IL, USA) was used to determine the optical density at 450 nm. Each experiment was performed with 3 independent replications.
4
Serum PGE2 Quantification in Rats
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Blood was collected from the right femoral artery of rats and centrifuged at 1,000 x g for 10 min at room temperature to obtain blood serum. The concentration of serum PGE2 was quantified using an ELISA Kit for PGE2 (cat. no. CEA538Ge; Wuhan USCN Business Co., Ltd.) according to the manufacturer's instructions. Plates were read using a Thermo microplate reader (Thermo Fisher Scientific, Inc.) at a wavelength of 450 nm.
5
Assessing Cytotoxicity of TSZ in SH-SY5Y Cells
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The inhibitory effect of TSZ [20 ng/mL TNF-ɑ (human), 100 nM Smac mimetics and 20 μM Z-VAD(OH)-FMK] on SH-SY5Y cells was assessed by CCK-8 assay (Cell Counting Kit-8, DOJINDO, Japan) according to the manufacturer’s protocol. Cells (1.5 × 103 cells/mL) were placed in 96-well plates and treated with TSZ for 10 h in the incubator. After intervention in different groups, 100 μL of CCK-8 dilution solution was added to each group, and cells were cultured at 37 °C for 2 h. Absorbance was measured at 450 nm using a Thermo Microplate Reader (Thermo, USA).
6
Cell Viability Assay of Celecoxib
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The cell viability was determined by using CCK8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instruction. Bel7402 and HepG2 cells were seeded at a density of 104 cells/well in 96-well plates for 24 hrs. Then the cells were treated with serial concentration (0, 10, 30, 50, and 70 μM) of celecoxib for 24 and 48 hrs. A 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for 1 hrs in the dark. The optical density of each well was measured at 450 nm using the Thermo microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
7
Cell Viability Assay with MCC950
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HCT116 cells were seeded at a density of 104 cells/well in 96-well plates for 24 h. Then the cells were treated with DMSO or MCC950 (2 μM) for 24 h. Ten microliters of CCK8 solution (Dojindo, Kumamoto, Japan) was then added and incubated at 37°C for 1 h in the dark. The optical density was measured at 450 nm using the Thermo microplate reader (Thermo Fisher Scientific, Waltham, MA, U.S.A.).
8
Cell Viability Assessment via MTT Assay
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MTT assay was used to determine cell viability after IR treatment. Cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After treatment, the cells were incubated with 1 mg/mL MTT solution for a 4-hr incubation. After removing the media, the formazan products were solubilized with 200 μL dimethyl sulfoxide. Cell viability was assessed by measuring absorbance at 562 nm using a Thermo microplate reader (Thermo Fisher Scientific, USA).
9
Cell Viability Assay in 96-well Plate
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The cells were seeded in a 96‐well plate to the density of 50%‐70% confluence. CCK8 assay was performed according to the instruction (Dojindo). The optical density was then measured by a Thermo microplate reader (Thermo Fisher Scientific) at 450 nm.
10
Cell Viability Assay with PA and TSF
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Cells were plated into 96-well plates and incubated for 24 h to allow cell adherence. Then, the culture medium was replaced with DMEM supplemented with PA (0.3 mM) and TSF (0, 25, 50, 100, 200, and 400 μg/mL). After 24 h of incubation, cell viability was determined by colorimetry using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Insoluble formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and measured at 490 nm with a Thermo microplate reader (Thermo Fisher Scientific, Waltham, MA, United States).
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