Ldh activity assay kit

Briefly, 1 × 10⁵ cells were seeded in a 6‐well plate. Culture medium was changed to fresh DMEM after incubation overnight. After 48 hours of transfection, the cell culture supernatant was collected and the concentration of lactate and glucose as well as the activity of LDH and pyruvate dehydrogenase (PDH) were assayed using a lactate assay kit (Jiancheng Bioengineering, Nanjing, China), glucose assay kit (Jiancheng Bioengineering), LDH activity assay kit (Jiancheng Bioengineering), and PDH activity assay kit (Jiancheng Bioengineering) according to the manufacturer's protocol, respectively. As for ATP concentration, cells were collected and lysed with ATP lysis buffer after 48 hours of transfection, and ATP concentration was measured using an ATP assay kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer's protocol. Values were normalized to the protein content of the cell lysate and expressed as a fold increase over vector control.

To measure the LDH activity in cells, five parallel wells were set for each experimental group and infected with Bag-1 or negative control siRNA for 48 hrs and then treated with 5 μg/mL cisplatin for an additional 24 hrs. Next, the culture medium was collected for detection of the LDH activity using an LDH activity assay kit (Nanjing Jian Cheng Biotechnology, Nanjing, People’s Republic of China) using the manufacturer’s protocol. Each sample was run in triplicate.
To assay the intracellular Ca²⁺ concentration, the duplicate cells were subjected to the Fluo-3/AM kit (Jiangsu Biyuntian Biological Technology Research Institute, Jiangsu, People’s Republic of China) analysis using the manufacturer’s protocol. Each sample was run in triplicate.

Cell culture medium was collected and LDH activity was measured using a commercial LDH Activity Assay kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. Briefly, 25 µl cell supernatant was mixed with 25 µl substrate in the kit and the solution was incubated at 37˚C for 15 min. A total of 25 µl 2,4-dinitrophenylhydrazine in the kit was then added into the samples. Following incubation at 37˚C for 15 min in the dark, 250 µl 0.4 mol/l NaOH solution was added and the mixture was further incubated at room temperature for 5 min. Absorbance was recorded at a wavelength of 450 nm using a microplate reader.