Tissue from the CA3 subregion was isolated for assaying. Tissues and cells were lysed by radioimmunoprecipitation assay lysis buffer, and total protein was extracted for western blotting experiments. The primary antibodies were NR2B (Cell Signaling Technology, 14544s) and glyceraldehyde phosphate dehydrogenase (Cell Signaling Technology, 5174s), and the secondary antibody was horseradish peroxidase-linked anti-rabbit immunoglobulin G (Cell Signaling Technology, 7074s). After 24 h of irradiation, the cell activity was detected using the MTS reagent kit (Promega, G3581).
Mts reagent kit
Manufactured by Promega
Sourced in United States
The MTS reagent kit is a colorimetric assay used to measure cell proliferation and cytotoxicity. It utilizes a tetrazolium compound that is reduced by viable cells, producing a colored formazan product that can be quantified spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Anti rabbit immunoglobulin g horseradish peroxidase linked, by Cell Signaling Technology (1 mentions)
Microplate reader, by Omega Bio-Tek (1 mentions)
Mcc950, by MedChemExpress (1 mentions)
Spectrophotometric microplate reader, by Agilent Technologies (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Cell death detection elisaplus kit, by Roche (1 mentions)
7 protocols using mts reagent kit
1
Western Blot Analysis of NR2B in CA3 Subregion
2
MTS Cell Proliferation Assay for A549 Cells
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The MTS cell proliferation assay was used to quantify viable A549 cells. In brief, A549 cells were seeded onto 96-well microplates (5×104 cells/well) in 100 µl culture medium (DMEM with 10% FBS) and cultured until the cells reached 70% confluency. Following this, cells were further cultured for 0, 12, 24 or 48 h, with medium containing DEX at a range of concentrations (0, 0.1, 1.0 and 10 mmol/l). The cell proliferation assay was performed using the MTS reagent kit (MTS; Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. In brief, 10 µl MTS was added, and cells were incubated at 37°C in a humidified incubator for 1–2 h. The absorbance values for each well were detected at 490 nM using a micro plate reader (Omega Bio-Tek Inc., Norcross, GA, USA).
3
Measuring Cell Viability and Apoptosis
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The reductive capacity of cells was measured either on INS-1 cells or dispersed human islet cells when the INS-1 cells or islets were subjected to 5 or 20 mM glucose for 72 h in the absence or presence of test agents or after down-regulation of VDAC1 and VDAC2 as described elsewhere. Measurement of reductive capacity was performed using the MTS reagent kit according to the manufacturer’s instructions (Promega) and (Janjic and Wollheim, 1992 (link)). Apoptosis was measured with the Cell Death Kit (Roche Diagnostics), which quantifies the appearance of cytosolic nucleosomes.
To measure the cell viability in living cells, we used EthD1 (Ethidium homodimer-1) and calcein to indicate death and live cell in INS-1 cells, respectively according to manufacturer (Thermo Fisher, USA). The plasma membrane targeted VDAC1 (plVDAC1) and mitochondrial VDAC1 (mtVDAC1) were overexpressed in INS-1 cells cultured with either 5 mM glucose (5G) or 20 mM glucose (20G). The mean intensity of Ethidium and Calcein were calculated to indicate live and death cells, respectively.
To measure the cell viability in living cells, we used EthD1 (Ethidium homodimer-1) and calcein to indicate death and live cell in INS-1 cells, respectively according to manufacturer (Thermo Fisher, USA). The plasma membrane targeted VDAC1 (plVDAC1) and mitochondrial VDAC1 (mtVDAC1) were overexpressed in INS-1 cells cultured with either 5 mM glucose (5G) or 20 mM glucose (20G). The mean intensity of Ethidium and Calcein were calculated to indicate live and death cells, respectively.
4
Measuring Cell Reductive Capacity
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The reductive capacity of cells was measured on INS-1 cells when the INS-1 cells were subjected to 5 or 20 mM glucose for 72 h in the absence or presence of test agents or after downregulation of Gpr142. Measurement of reductive capacity was performed using the MTS reagent kit (Cat# G3580) according to the manufacturer’s instructions (Promega). Apoptosis was measured with the Cell Death Kit (Roche Diagnostics), which quantifies the appearance of cytosolic nucleosomes.
5
A498 Cell Culture and Analysis
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A498 cells were purchased from the American Type Culture Collection. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. The medium was changed every 2 days. Cells were used for experiments in their logarithmic growth phase. The MTS reagent kit was obtained from Promega Corporation. Primary antibodies for NLRP3 (cat. no. ab263899; 1:1,000), caspase-1 (cat. no. ab207802; 1:1,000), IL-1β (cat. no. ab216995; 1:1,000), cleaved IL-1β p15 (cat. no. ab33774; 1:1,000), MMP-2 (cat. no. ab181286; 1:1,000) and GAPDH (cat. no. ab181602; 1:1,000) were purchased from Abcam. Secondary HRP-conjugated goat anti-rabbit IgG antibodies were purchased from Beyotime Company, Shanghai, China. (cat. no. A0208; 1:10,000). UA was purchased from Tianjin Jinyao Amino Acid Co., Ltd., and MCC950 was purchased from MedChemExpress.
6
Dapagliflozin Effects on C2C12 Cell Proliferation
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C2C12 cells were treated with the indicated doses of dapagliflozin for 24 h in the hyperglycemic condition (final concentration of glucose: 25 mM). Following the treatment, cells were reseeded in a 96-well plate and exposed to hypoxia at indicated times. Cell proliferation assay was performed by using an MTS reagent kit (Promega, Madison, WI) for 2 h. Viable cell numbers were measured with a spectrophotometric microplate reader (BioTek Instruments, Winooski, VT) at a wavelength of 490 nm. For crystal violet staining, C2C12 cells were seeded in a 24-well plate and treated with indicated doses of dapagliflozin for 24 h. Following 24 h exposure to hypoxia, cells were fixed by 5 min incubation with 4% paraformaldehyde (PFA) and stained for 30 min with 0.05% crystal violet (Beyotime, Shanghai, China).
7
Gprc5c Knockdown Islet Apoptosis
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After treatment with Gprc5c shRNA lentiviral particles (see above), mouse islets were dispersed into single cells using Ca 2+ free medium. The islet cells were then cultured with or without a cocktail of pro-apoptotic cytokines (IL-1β, 100 ng/mL; TNFα, 125 ng/mL; and INFγ, 125 ng/mL) for 24 h in RPMI 1640 with 5 mmol/L glucose and 10% FSB supplemented with 5 μmol/L ATRA. Measurements of cell viability and detection of apoptosis were performed using the MTS reagent kit (Promega) and Cell Death Detection ELISA plus kit (Roche) according to the manufacturer's instructions.
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