Ldh activity colorimetric assay kit

For the determination of ATP levels, the amount of ATP was measured with an ATP Detection Assay Kit (Cayman, 700410) according to the assay protocol. For the assay of cellular glycolysis, glycolytic activity was measured by using Glycolysis Cell‐Based Assay Kit (Cayman, 600450), which allows for colorimetric detection of L‐lactate, the end product of glycolysis, produced and secreted by cultured cells. For the measurement of the cellular glucose uptake, Glucose Uptake Cell‐Based Assay Kit (Cayman, 600470) was employed for measuring the cellular glucose uptake. This kit employs 2‐Deoxy‐2‐[(7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl)amino]‐D‐glucose (2‐NBDG), a fluorescently labelled deoxyglucose analogue, as a probe for the detection of glucose taken up by cultured cells. The level of cellular glutamine was determined with Glutamine Colorimetric Assay Kit (BioVision, K556‐100). Cellular α‐ketoglutarate assay was detected by using Ketoglutarate Colorimetric/Fluorometric Assay Kit (BioVision, K677‐100) according to the assay protocol. To measure the activity of lactate dehydrogenase (LDH), LDH Activity Colorimetric Assay Kit (BioVision, K726‐500) was utilised to quantify LDH activity in cells.

Caco-2 cells were grown in DMEM (Corning, USA) medium containing L-glutamine, 10% FBS, and 1% antibiotic–antimycotic solution^{33 (link)}. Caco-2 cells were seeded at a concentration of 1.0 × 10⁴ cells in a 96-well tissue culture plate and incubated in 5% CO₂ at 37 °C. After 5 days at late post-confluence, Caco-2 cells were treated with overnight-cultured LAB isolates at a concentration of approximately 1.0 × 10⁸ cells and incubated in 5% CO₂ at 37 °C. After 24 h incubation, the culture medium was centrifuged for 5 min at 3000 rpm and the supernatants were analysed for LDH measurement using LDH Activity Colorimetric Assay Kit (BioVision, USA)^{81 (link)}. The lysate extracted from Caco-2 cells at late post-confluence using 0.1% Triton-X100 (Sigma-Aldrich, USA) was used as the positive control. The culture medium of Caco-2 cells at late post-confluence without LAB treatment was used as the negative control. Cytotoxicity (%) was calculated by (test sample – negative control)/(positive control – negative control) × 100.

An LDH Activity Colorimetric Assay Kit (Biovision, USA) was used. Briefly, 1 × 10⁶ cells were homogenized with ice-cold assay buffer, incubated on ice for 10 min, and centrifuged at 10,000× g for 15 min. Next, the supernatant was transferred to a fresh tube, and 100 μL of the reaction mixture (10 μL sample, 40 μL assay buffer, and 50 μL reaction mix) was transferred to a 96-well plate. Absorbance was immediately measured at 450 nm in kinetic mode at 37 °C for 0–20 min. LDH activity was measured based on the change in absorption during a specific time frame.