For glucose uptake, mLPS1 and mLPS1ΔNICD cells were seeded in 24-well plates at a density of 1 × 105 cells/well and co-cultured in RPMI 1640 medium supplemented with 10% FBS for 16 h. The cells were then washed with PBS and cultured for 6 h in fresh RPMI 1640 medium. The culture medium was collected, and glucose uptake was analyzed using a OneTouch Ultra 2 Blood Glucose Meter (Lifescan). For the lactate production and ATP assays, the lactate production was analyzed using the L-Lactate Assay Kit II (Eton Bioscience) according to the manufacturer’s instructions. Simultaneously, cell lysates were collected to measure ATP production using an ATP Detection Assay Kit (Cayman, 700410), following the manufacturer’s instructions.
L lactate assay kit 2
Manufactured by Eton Bioscience
Sourced in United States
The L-Lactate Assay Kit II is a colorimetric assay designed to quantify L-lactate levels in various samples. The kit utilizes an enzymatic reaction that produces a colored product, which can be measured using a spectrophotometer. The assay is ready-to-use and provides a simple, accurate, and high-throughput method for L-lactate determination.
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6 protocols using l lactate assay kit 2
1
Glucose Uptake and Metabolic Assays in mLPS1 Cells
2
Quantitative Lactate Measurement
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The l -Lactate Assay Kit II (1200051002, Eton Bioscience) was employed to quantitate the amount of lactate.
3
Measuring Metabolic Activity in DLBCL Cells
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The Lactate dehydrogenase (LDH) assay kit (C0016, Beyotime Biotechnology) and L-Lactate Assay Kit II (1200051002, Eton Bioscience) were used to measure intracellular LDH and lactate levels, respectively, in DLBCL cells according to the manufacturer’s protocols. ATP generation was detected using an ATP detection assay kit (S0026, Beyotime Biotechnology).
4
L-Lactate Quantification Protocol
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The analysis was performed using a L-Lactate Assay Kit II (Eton Bioscience, San Diego, CA, US) following the manufacturer's instructions (see also Supplementary Materials and Methods ). The standard curve was made using a L-lactate standard provided in the kit and the measure of the absorbance was performed at 570 nm in a microplate reader (Sunrise Tecan). The calculations were made correcting for the background by subtracting the value of the blank from all sample readings.
5
Measurement of Cellular Lactate Production
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The L-Lactate assay kit II (Eton Bioscience #1200052002) was used for lactate measurements. Cells were seeded (15,000 cells/well) in a 96 well plate in triplicate, media without cells was added in six wells to use as a blank. The cells were cultured overnight for 16 hrs, then starved for 4 hrs in DMEM (A14430-01) supplemented only with 1% P/S, L-glutamine (2 mM) and glucose (25 mM). The cells were then pretreated with α-c yano-4-h ydroxyc innamic acid (CHC) and then stimulated with HRG for 0, 1, 4, 8, and 16 hrs. The media was taken at each corresponding time point and frozen. The samples were then thawed and diluted 20-fold in PBS. The samples were incubated for 30 min in the assay kit’s L-lactate detection solution and read at 570 nm. Significance was assessed with the Student’s t-test.
6
Measurement of Cellular Lactate Levels
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The levels of Lactate concentration was measured using the L-Lactate Assay Kit II (Eton Bioscience, 200051002). Brie y, the cell culture medium was collected and the Sample Mix was prepared using the L-Lactate Assay Buffer (1x). Then the Reaction Solution was prepared using L-Lactate Assay Buffer, L-Lactate Assay Enzyme Mix and Assay Probe. After incubating the Sample Mix with the Reaction Solution for 30 min at 37 °C , the absorbance at OD 570 nm was measured using the microplate reader and then the L-Lactate concentration was calculated.
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