We used lactate dehydrogenase (LDH) to evaluate cell damage. According to the manufacturer’s manual, the commercial Lactate Dehydrogenase Kit (Roche, Mannheim, Germany) was used to measure the lactate dehydrogenase in the media. The cell viability of the H9C2 cells was detected by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay22 . In brief, differently treated H9C2 cells were seeded into 96‐well plates and incubated with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide solution (1 mg/mL; Sigma‐Aldrich, St. Louis, MO, USA) at 37°C for 4 h. We added dimethyl sulfoxide (100 mL/well) to resolve the formazan crystal. The absorbance was measured at 570 nm by Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA).
Lactate dehydrogenase kit
Manufactured by Roche
Sourced in Germany
The Lactate Dehydrogenase Kit is a laboratory diagnostic tool used for the quantitative determination of lactate dehydrogenase (LDH) activity in various biological samples, such as serum, plasma, and other body fluids. LDH is an enzyme involved in the conversion of lactate to pyruvate, and its measurement can provide insights into the overall metabolic state of the body.
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3 protocols using lactate dehydrogenase kit
1
Evaluating H9C2 Cell Viability
2
Cytotoxicity Assessment in Cell Lines
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A lactate dehydrogenase kit (Roche, Basel, Switzerland) was used to assess cytotoxicity. At the end of the experiments, for FHL124 cells 50 µL of the culture medium of FHL124 cells seeded at a density of 50,000 cells per culture dish was sampled and transferred to a clear 96-well plate already containing 150 µL EMEM per well. For human lens epithelium, 100 µL of culture medium from a whole capsulorhexis stabilized by pins in a culture dish and 100 µL EMEM medium were used instead. A working solution was made following the manufacturer's instruction, and 100 µL of the working solution was then added into each well. The whole plate was then covered in foil and incubated on a shaker at room temperature for 15 minutes before being measured with the FLUOstar Omega microplate reader at 490 nm.
3
Cytokine and Galectin-3 Stimulation Assay
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At the day of experiments, the cell medium was replaced by pre-warmed 500 μL DMEM/antibiotics per well. Cells were stimulated with LPS (10 ng/mL), TNF-α (1, 20, 100 ng/mL), IL-1β (1, 20, 100 ng/mL), galectin-3 (5, 50, or 100 μg/mL), or the combinations of LPS (10 ng/mL) or TNF-α (100 ng/mL) with galectin-3 (5 or 100 μg/mL) for 24 h. Cell culture media from each well was collected and stored at -80 ° C until analysis. Each experiment was repeated at 3 independent times using 6 animals/repeat. Vehicle and treatment wells were situated on the same plate, and 2 samples for each treatment were analyzed for every repeat.
To avoid that analyses were affected by varying cell counts, cells were collected in RIPA buffer for protein concentration test (BCA assay kit), and all results presented were corrected for protein content. Cytotoxicity of the different treatments was tested by assessing LDH release into the supernatant (Lactate Dehydrogenase Kit; Roche) according to the manufacturer's recommendation.
To avoid that analyses were affected by varying cell counts, cells were collected in RIPA buffer for protein concentration test (BCA assay kit), and all results presented were corrected for protein content. Cytotoxicity of the different treatments was tested by assessing LDH release into the supernatant (Lactate Dehydrogenase Kit; Roche) according to the manufacturer's recommendation.
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