After 7 days of cultivation, pseudo-islets were preincubated for 3 h at 37 °C with the cell culture medium containing 3.3 mmol/L glucose. Subsequently, the cells were incubated with fresh medium containing 3.3 mmol/L glucose for 1 h. Then, the medium was replaced with medium containing 20 mmol/L glucose, and the cells were incubated for 1 h. At the end of each step, the medium was collected for determination of the insulin level. The concentration of insulin in the medium was measured by the Mercodia Rat Insulin ELISA Kit (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions. The relative cell viability of the islet cells was determined by a WST-8 (water-soluble tetrazolium salt) assay using Cell Count Reagent SF kit (Nacalai Tesque, Kyoto, Japan). In addition, the DNA content of the pseudo-islets was determined by a DNA quantitation kit (Bio-Rad, Hercules, CA). The values of insulin secretion were normalized to the absorbance values from WST-8 assay or the DNA content in the islet cells.
Cell count reagent sf kit
Manufactured by Nacalai Tesque
Sourced in Japan
The Cell Count Reagent SF kit is a laboratory product designed for determining the cell count in a sample. It provides a simple and accurate method for counting cells, but does not include any interpretation or recommendations for its intended use.
Automatically generated - may contain errors
5 protocols using cell count reagent sf kit
1
Insulin Secretion Dynamics of Pseudo-Islets
2
Cell Proliferation Assay for ATDC5 and MC3T3-E1 Cells
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Cell proliferation activity was measured using a colorimetric Cell Count Reagent SF kit (Nacalai Tesque) according to manufacturer's instruction. Cells were plated in 96-well plates at a density of 3,000 cells/well (ATDC5) or 10,000 cells/well (MC3T3-E1). Cells were treated with BSA or AGE-BSA for 2 days. After cells were incubated with WST-8 for 2 hours, proliferative activities were measured on a microplate reader at 450 nm (model680, Bio-Rad, Tokyo, Japan). There was no difference in the number of dead cells between the cell lines determined by a trypan blue exclusion assay.
3
Cell Viability Assay for HLE Cells
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HLE cells with or without added H2O2 and ferulic acid treatments were incubated in 96-well microplates (Iwaki, Chiba, Japan). The viability of the HLE cells was calculated by the Cell Count Reagent SF kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's instructions. The absorbance at 450 nm was measured, and the cell viability (%) was represented as the percentage of the absorbance measured for unstimulated HLE cells for each point (normal cells).
4
UV-induced Cytotoxicity Assay in HaCaT Cells
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The culture medium of HaCaT cells seeded in a 96-well plate was replaced to PBS, and cells were exposed to UVB radiation (15 or 30 mJ). Cells were cultured in DMEM medium containing 10% FBS for 24 h. To assess cell viability, cells were estimated with a Cell Count Reagent SF kit (Nacalai Tesque). For cytotoxicity assay, culture medium (DMEM) was replaced to PBS, and collected PBS after incubation for 2 h. Cytotoxicity was determined by Cytotoxicity LDH assay kit according to manufacturer's instructions (WAKO).
5
Evaluating PQQ Effects on Fibroblast Metabolism
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NIH/3T3 fibroblasts were seeded into a 96-well culture plate and cultured until they reached 70–80% confluence. The cells were washed with serum- and pyruvate-free DMEM and then starved in the medium. Twenty-four hours later, the medium was exchanged with fresh serum- and pyruvate-free medium, and then the cells were exposed to PQQ. After 24 h of incubation, lactate levels in the culture media were determined using a lactate assay kit (Kyowa Medex, Tokyo, Japan) as described by the manufacturer’s instructions. Cellular ATP levels were determined using an ATP luminescence kit (TOYO B-Net, Tokyo, Japan) according to the manufacturer’s instructions. Lactate and cellular ATP levels were normalized to cell number. Cell number was determined by WST-8 reduction assay using a Cell Count Reagent SF kit (Nacalai Tesque) according to the manufacturer’s instructions.
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