Metabolites were dissolved at 90°C in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) at various concentrations ranging from 2 to 10 mg/ml, followed by gradual cooling of the solution. SH-SY5Y cells were seeded at 2 × 105 per well in six-well plates and were allowed to adhere overnight, followed by incubation with medium containing metabolites for 24 hours. Control cells were incubated with medium that was treated in the same manner without any addition of metabolites. The apoptotic effect was evaluated using the MEBCYTO Apoptosis kit (MBL International), according to the manufacturer’s instructions. Briefly, the adherent cells were trypsinized, detached, and combined with floating cells from the original growth medium. They were then centrifuged and washed once with PBS and once with binding buffer. Cells were incubated with annexin V–FITC and PI for 15 min in the dark, then resuspended in 400 μl of binding buffer, and analyzed by flow cytometry using a single laser emitting excitation light at 488 nm. Data from at least 104 cells were acquired using BD FACSort and the CellQuest software (BD Biosciences). Analyses were done with FlowJo software (TreeStar, version 14). Each experiment was repeated three times.
Dmem nutrient mixture f12 ham s
Manufactured by Sartorius
Sourced in Israel
DMEM/Nutrient Mixture F12 (Ham's) is a cell culture medium designed for the growth and maintenance of various mammalian cell lines. It is a balanced salt solution that provides the necessary nutrients, amino acids, vitamins, and other components to support cell proliferation and survival in vitro.
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6 protocols using dmem nutrient mixture f12 ham s
1
Metabolite-Induced Apoptosis Assay
2
Oxalate Cytotoxicity Screening in Cell Lines
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ARPE-19 cells and HEK-293 cells (2 × 105 cells/ml) were cultured in 96-well tissue microplates (100 µl per well) and allowed to adhere overnight at 37 °C. Oxalate assemblies were formed in vitro as outlined above, except the solvent was DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries, Israel) without FBS. The concentrations of all oxalate preparations were approximately 6 mM. Only half of each plate was seeded with cells. Medium with no oxalate, which was treated in the same manner, was used as a negative control. Medium (100 µl) with or without oxalate assemblies was added to each well. After overnight incubation at 37 °C, cell viability was evaluated using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide MTT cell proliferation assay (reagent purchased from Sigma, Rehovot, Israel), according to the manufacturer’s instructions. Briefly, 10 µl of 5 mg/ml MTT reagent dissolved in PBS was added to each of the 96 wells, followed by a 4 h incubation at 37 °C. Then, 100 µl of extraction buffer (20% SDS dissolved in a solution of 50% dimethylformamide and 50% DDW) was added to each well, followed by 30 min incubation at 37 °C in the dark. Finally, color intensity was measured using an ELISA plate reader at 570 nm and background subtraction at 650 nm. The results represent three biological repeats; the data are presented as mean ± SD.
3
Cell Viability Assessment of Tyrosine Assemblies
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SH-SY5Y cells (2 × 105 cells/mL) were cultured in 96-well tissue microplates (100 µL per well) and allowed to adhere overnight at 37 °C. Tyr assemblies were formed in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) at a concentration of 2 mg/mL, as outlined above. Only half of each plate was seeded with cells, with the other half used as a blank control. Medium with no Tyr, which was treated in the same manner, served as a negative control, represented by zero. Medium (100 µL) with or without assemblies was added to each well. After overnight incubation at 37 °C, cell viability was evaluated using 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide MTT cell proliferation assay (Sigma-Aldrich, Rehovot, Israel) according to the manufacturer’s instructions. Briefly, after overnight incubation at 37 °C with the metabolite, 10 µL of 5 mg/mL MTT reagent dissolved in PBS was added to each of the 96 wells, followed by 4 h of incubation at 37 °C. Next, 100 µL extraction buffer (50%DMF, 20%SDS in DDW) was added to the wells, followed by 30 min incubation at 37 °C in the dark. Finally, color intensity was measured using an ELISA plate reader at 570 nm and background subtraction at 680 nm. The results represent three biological repeats. Values are means ± SD, student’s t-test, ** p < 0.001.
4
Cytotoxicity Evaluation of Test Compounds
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Test compounds and the negative control (PBS) were dissolved in PBS at 1 mg/ml and heated for 50 min at 90 °C, followed by gradual cooling of the solution overnight. Samples were then diluted two fold in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) lacking fetal bovine serum. 1% TritonX-100 in PBS was used as a positive control. 2 × 105 cells/ml of the HaCat and HEK 293 cell lines were cultured in 96-well tissue microplates (100 µl per well) and were allowed to adhere overnight at 37 °C. Cells were plated on only half of the microplate with the other half consisting of growth medium with and without each of the test compounds. Cell viability was evaluated using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay following incubation with the test and control compounds overnight at 37 °C. Briefly, 10 µl of 5 mg/ml MTT dissolved in PBS was added to each well and incubated for 4 h at 37 °C. This was followed by the addition of 100 µl of the extraction buffer [20% SDS dissolved in a solution of 50% dimethylformamide and 50% DDW (pH 4.7)] to each well, and incubation at 37 °C for 30 min. The resulting color intensity was then measured using a microplate reader at 570 nm. The results presented are the mean of three independent experiments conducted ± the standard error of the mean.
5
Cell Viability Assay of Metabolites
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SH-SY5Y cell line (2 × 105 cells/ml) were cultured in 96-well tissue microplates (100 μl per well) and allowed to adhere overnight at 37°C. Metabolites were dissolved at 90°C in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) at various concentrations ranging from 0.2 to 10 mg/ml, followed by gradual cooling of the solution. Each plate was divided, and only half of it was plated with cells. The negative control, represented by zero, was prepared as medium with no metabolites, which was treated in the same manner. Medium (100 μl) with or without metabolites was added to each well. After incubation for 6 hours at 37°C, cell viability was evaluated using the XTT cell proliferation assay kit (Biological Industries) according to the manufacturer’s instructions. Briefly, 100 μl of the activation reagent was added to 5 ml of the XTT reagent, followed by the addition of 100 μl of activated XTT solution to each well. After 2.5 hours of incubation at 37°C, color intensity was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader at 450 and 630 nm. Results are presented as means ± SEM. Each experiment was repeated three times.
6
Metabolite Solubility and Cell Viability
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Metabolites were dissolved at 90°C in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) at various concentrations ranging from 2 to 10 mg/ml, followed by gradual cooling of the solution. Next, metabolite samples were centrifuged at 20,000g for 1 hour at 4°C. The control reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with the resulting supernatant medium that contained the soluble form of the metabolites and were further analyzed using XTT cell viability assay and annexin V–FITC and PI apoptosis assay as was previously presented.
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