D lactic acid l lactic acid kit

DU145 and 22Rv1 transfectants were incubated in Opti-MEM (without phenol red, Gibco) for 16 hours 37°C 5% CO₂. The lactic acid concentration of the supernatant was determined with the D-Lactic acid/L-lactic acid kit (R-Biopharm) according to the manufacturer's instruction, and was then normalized to the protein concentration of each sample. The ratio mock:COMP represents the increase of lactic acid in COMP-transfected cells as compared to mock-transfected cells.

Aliquots of CVF were prepared for biochemical characterization by diluting native CVF samples 50-fold with unbuffered 0.9% saline solution, gentle vortex mixing, and centrifuging at 1,000 × g for 2 min to collect “diluted CVF cell-free supernatant,” which was then stored at −80°C. Prior to use, aliquots were thawed, and serial dilutions were prepared with unbuffered 0.9% saline solution within the linear range of each assay. Lactic acid levels were measured using a d-lactic acid/l-lactic acid kit (R-Biopharm, Darmstadt, Germany) following the manufacturer’s protocol adapted to a 96-well format. This method measures the concentration of lactate anion after neutralization of the sample to pH 7 to 9 to calculate the total concentration of lactic acid in the sample. At pH levels of <7, samples contain the lactate anion and protonated lactic acid, the sum of which we refer to as lactic acid. The protonated form of lactic acid present in CVF samples was calculated from the sample pH and lactic acid concentration using the Henderson-Hasselbalch equation as previously described (22 (link), 35 (link)). For these calculations, we made the assumption that the pK_a value for lactic acid in CVF is 3.86. The biochemical characteristics of CVF samples are summarized in Table S2.

The ruminal fluid used for lactate analysis was first boiled at 100 °C for 10 min and then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatants were collected and used for lactate analysis. The UV method was used for the determination of d- and l-lactic acid concentrations by using the d-lactic acid/l-lactic acid kit from R-Biopharm (R-Biopharm AG, Darmstadt, Germany). The analysis procedure was adapted from the manufacturer’s protocol. Specifically, 20 µL of sample, 80 µL of deionized water, 100 µL mix of glycylglycine buffer and l-glutamic acid, 20 µL of NAD, and 2 µL of glutamate-pyruvate transaminase suspension were loaded in a 96-well UV microplate, incubated at room temperature (RT) for 5 min, and read at 340 nm on a microplate spectrophotometer (Spectra Max 340 PC, Molecular Devices Corporation, Sunnyvale, CA, USA). Then, 2 µL of d-lactate dehydrogenase solution was added to each well and incubated at RT for 180 min and absorbance was read to determine the concentration of d-lactic acid. Then 2 µL of l-lactate dehydrogenase solution was added to each well and incubated in RT for another 180 min, and absorbance was read to determine the concentration of l-lactic acid. A 99% recovery of spiked concentration for both d- and l-lactic acid was considered as non-inhibitory dilution.

OTC gels analyzed in this study are listed in Table 1. The osmolality of gels was measured in triplicate using a cryoscopic osmometer (Gonotec Osmomat 030-D, Gemini BV, Apeldoorn, Netherlands) calibrated at 100, 300, and 2,000 mOsmol/kg. Gels which initially gave values above the upper limit of quantitation (3,000 mOsmol/kg) were diluted 1:2 in H₂O and measured again, with the reassessed values expressed as an extrapolated value. Gel pH was measured using a pH electrode connected to a pH meter (TPS-Aqua meter and IJ sensor, Vintessential Laboratories, Orange, Australia) which allows accurate pH measurement of viscous fluids. LA levels were measured using the D-lactic acid/L-lactic acid kit (R-Biopharm, Darmstadt, Germany) as previously described (11 (link)). This method measures the concentration of D- and L-lactate anions after neutralization of the sample to pH 8–10 to calculate the total lactate concentration in the sample. The concentration of protonated LA present in undiluted gels was then calculated using the sample pH and the Henderson-Hasselbalch equation and a pKa value for LA of 3.86 as previously described (6 (link)).