Lactate assay kit

mES cells were cultured for 24 h and then changed into fresh ES cell culture medium. Eight hours later, the glucose levels in the culture medium were measured using the Glucose (GO) Assay Kit (Sigma), and the lactate levels were determined using a Lactate Assay Kit (Eton Bioscience).

Cells were cultured on 96-well plate for 3 days and the supernatant was collected. Glucose consumption was analyzed using glucose assay kit (Eton Bioscience), according to manufacturer’s protocol. Lactate production was analyzed using lactate assay kit (Eton Bioscience), followed the manufacturer’s protocol.

The lactate released by parasite oocysts and sporozoites was detected using a lactate assay kit (Eton Bioscience Inc., San Diego, CA). In this assay, LDH converts lactate and NAD⁺ into pyruvate and NADH, and then a NADH-coupled enzyme reaction reduces a tetrazolium salt INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride) into formazan that exhibits an absorbance maximum at 490 nm. Oocysts (3×10⁷) removed from refrigeration (4°C) were washed twice with PBS and resuspended in 200 μL PBS. Free sporozoites were prepared from 3×10⁷ oocysts by an excystation procedure, as described above, and resuspended in 200 μL PBS. Both oocyst and sporozoite samples were subjected to centrifugation after incubation at 37°C for 1 and 4 h, respectively, from which 20 μL of supernatants were used for detecting lactate contents. The assay was carried out in 70 μL of reaction buffer in 384-well microplates. The reactions were initialized by adding 50 μL of lactate reaction solution into 20 μL sample solutions or lactate standards (20 to 640 μM). After incubation at 37°C for 1 h, the reactions were stopped by adding 50 μL of 0.5 M acetic acid. OD₄₉₀ values were measured using a Multiskan spectrum spectrophotometer (Thermo Scientific, West Palm Beach, FL).

pH of culture media was measured using a pH meter (Accumet AB15 Basic and BioBasic pH/mV/°C meter, Fisher Scientific). Lactate in culture media was measured using a lactate assay kit (Eton Bioscience, Inc.) and microplate reader (absorbance 490 nm, SpectraMax Plus⁵⁸⁴, Molecular Devices) in a quantitative manner with lactate standards. Lactate production was standardized per 10⁵ cells.

The lactate levels were measured using the Lactate Assay Kit (Eton Bioscience Inc., San Diego, CA, USA) and according to the manufacturer’s protocol. MCF7-H and MCF7-J cybrids were plated at 10,000 cells/well in 96-well plates, incubated overnight and lactate concentrations were analyzed. The solutions were diluted 1:2 and 1:4 to verify the repeatability of the assay. Standards and samples were set up as duplicates and quadruplicates and experiments were repeated twice.

Skin fibroblasts were seeded at 40,000 cells per well in a 96-well clear plate cultured in 200 μL of MEM containing 10% FBS for 24 hours or in MEM lacking FBS for 24 and 48 hours at 37 °C. Lactate concentration in the media was determined using the Lactate Assay Kit (Eton Bioscience Inc., San Diego, CA, USA, cat. # 120001100A). Briefly, 50 μL of media was mixed with 50 μL Lactate Assay Solution and incubated in the dark in a non-CO₂ incubator at 37 °C for 30 minutes. The reaction was quenched by addition of 50 μL of 0.5 M acetic acid. Samples were measured on a Spectra Max M3 plate reader using absorbance 490 nm. A standard curve containing L-lactate was generated for each assay. Cells were rinsed with PBS and incubated with 20 μL of RIPA lysis buffer for 10 minutes. Protein lysate concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, cat. # 23225). Lactate concentrations were normalized to total protein content and sample volume (200 μL). Each skin fibroblast was analyzed in triplicate wells. All technical and biological replicates were averaged for analysis.