Cells were seeded at 50 × 104 cells per well in a six-well plate. After 48 h, the pyruvate levels in the cells were determined using the Pyruvate Assay Kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. The pyruvate levels were then normalized to the cell number.
Pyruvate assay kit
Manufactured by Solarbio
Sourced in China
The Pyruvate Assay Kit is a colorimetric detection kit designed to quantify pyruvate levels in various sample types. It provides a simple and straightforward method to measure pyruvate concentrations.
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Lactic acid assay kit, by Solarbio (1 mentions)
Bc2205, by Solarbio (1 mentions)
Glucose assay kit, by Beyotime (1 mentions)
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Ldh cytotoxicity detection kit, by Beyotime (1 mentions)
Atp determination kit, by Beyotime (1 mentions)
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12 protocols using pyruvate assay kit
1
Pyruvate Assay in Cultured Cells
2
Intracellular Pyruvate and Extracellular Lactate Quantification
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The intracellular content of pyruvate, as well as lactate levels in the cell supernatant, were assessed using the Pyruvate Assay Kit (BC2205, Solarbio) and Lactate Assay Kit (BC2230, Solarbio) following the manufacturer’s recommended protocol. Briefly, cells from 6-well plates were collected into centrifuge tubes, and commercial extracts were added before sonication. Following centrifugation, the resulting supernatant was collected and used for subsequent analysis. For lactic acid, the cell supernatant was collected after 4 h of incubation for subsequent analysis. The absorbance value of the sample was then substituted into the generated standard curve to determine the corresponding index measurement. All obtained data were further normalized to the corresponding protein concentration of the cell extracts.
3
Quantifying Glioma Metabolic Markers
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Pyruvate assay kit (BC2205, Solarbio, China), lactate assay kit (BC2235, Solarbio, China), and ATP assay kit (BC0300, Solarbio, China) were used to measure the intracellular levels of pyruvate, lactate, and ATP, respectively. Glioma cells were treated with 0.3% DMSO, 30 μM P7C3, or 50 μM P7C3 for 24 hours. After that, an equal number of cells (5 × 106 cells from each sample) were collected for the subsequent measuring procedure according to the product manual. Relative absorbance was measured under a spectrophotometer (1510, Thermo Fisher, USA).
4
Metabolic Profiling of Shrimp Hemocytes and Intestine
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The Pyruvate (PA), ATP, and lactic acid (LA) assay kits (Solarbio Science and Technology) were used to detect the PA, ATP, and LA contents in hemocytes (cell number was counted using a hemacytometer) and intestine tissues of Ampkα-knockdown shrimp. The intracellular pyruvate content was detected via absorption at 520 nm using the pyruvate assay kit (Solarbio Science and Technology) according to the manufacturer’s instructions. The intracellular ATP content was detected via absorption at 340 nm using the ATP assay kit (Solarbio Science and Technology) following the manufacturer’s instructions. The lactic acid production in the culture supernatant was measured at 570 nm using a lactic acid assay kit (Solarbio Science and Technology) following manufacturer’s instructions.
5
Metabolite Quantification and Daptomycin Analysis
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The metabolites pyruvate and ATP were determined with the Pyruvate Assay Kit (Solarbio, BC2205) and ATP Assay Kit (Beyotime, S0026), respectively, according to the manufacturer’s instructions. Daptomycin was analyzed as described previously40 (link).
6
Serine Hydrolase Activity Assay
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The cell extract of E. ludwigii AA4 was prepared as above, except washing and resuspending in the extraction buffer (50 mM K2HPO4, 2.5 mM serine protease inhibitors) (Velayudhan et al., 2004 (link)). Three aliquots of 1 ml of cell extract containing total proteins of 10 mg/ml was incubated with 500 μl of 300 mM L -serine at 37°C for 6 min. Then, the concentration of pyruvate was determined by using a pyruvate assay kit (Solarbio, BC2205, China). The L -serine hydrolase activity was calculated as:
where c is the concentration of pyruvate in μg/ml, v is the total reaction volume in ml, M is the relative molecular mass of pyruvate, T is the reaction time in minutes, and m is the weight of cell extract in mg.
Two-tailed t-test (GrapPad Prism 8) was used for the statistical analysis. This experiment was repeated five times.
where c is the concentration of pyruvate in μg/ml, v is the total reaction volume in ml, M is the relative molecular mass of pyruvate, T is the reaction time in minutes, and m is the weight of cell extract in mg.
Two-tailed t-test (GrapPad Prism 8) was used for the statistical analysis. This experiment was repeated five times.
7
Quantifying Trehalose and Pyruvate Production in Fungi
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For each strain, three agar blocks containing mycelia (5 mm in diameter) were cultured in 25 ml YEPD medium with rotation (175 rpm), and the fresh mycelia were harvested after 3 days at 25 °C. Trehalase activity was measured with the Trehalase Assay Kit (Solarbio) according to the manufacturer's instructions, as was trehalose production (Li et al., 2019 ). For pyruvate production, three agar blocks containing mycelia (5 mm in diameter) were cultured in 20 ml GYEP with rotation (175 rpm). Trehalose production content was expressed as a ratio of trehalose production to fresh mycelial weight (mg/g). After 3 days at 28 °C, the fresh mycelia were harvested and pyruvate production was detected with the Pyruvate Assay Kit (Solarbio) according to the manufacturer's instructions (Li et al., 2019 ). Pyruvate production content was expressed as a ratio of pyruvate production to fresh mycelial weight (μg/g).
8
Metabolic Profile of TNBC Cells
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TNBC cells were incubated in phenol red‐free DMEM without FBS for 4 h. Next, a pyruvate assay kit (Solarbio), lactate assay kit (Solarbio) and ATP assay kit (Solarbio) were used to measure intracellular concentrations of pyruvate, lactate and ATP, respectively.
9
Transcriptomic Analysis of S. aureus Biofilm
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S. aureus biofilms were collected after different treatments and immediately stored in liquid nitrogen. The samples were detected for transcriptomic analysis with the mass spectrometry platform of OE Biotech (Shanghai, China). The data obtained were further analyzed using OE Cloud (OE Biotech) software.
qPCR was used to validate the expression levels of genes that were significantly regulated in the transcriptomics analysis results. Briefly, total S. aureus RNA was extracted from biofilms using a bacterial RNA extraction kit (Vazyme, China). cDNA was synthesized using a reverse transcription kit (EZBioscience, USA) after the determination of RNA concentration. cDNA obtained was subsequently amplified using SYBR Green Master Mix (EZBioscience, USA) and a LightCycler 480 (Roche, USA). The 16s RNA was selected as the housekeeping gene and the primers used were listed inTable S1 .
Finally, the glucose metabolism of S. aureus in biofilms was measured using the Glucose Assay Kit (Beyotime, China), Pyruvate Assay Kit (Solarbio, China), ATP Assay Kit (Beyotime, China), and NAD+/NADH Assay Kit (Beyotime, China), respectively.
qPCR was used to validate the expression levels of genes that were significantly regulated in the transcriptomics analysis results. Briefly, total S. aureus RNA was extracted from biofilms using a bacterial RNA extraction kit (Vazyme, China). cDNA was synthesized using a reverse transcription kit (EZBioscience, USA) after the determination of RNA concentration. cDNA obtained was subsequently amplified using SYBR Green Master Mix (EZBioscience, USA) and a LightCycler 480 (Roche, USA). The 16s RNA was selected as the housekeeping gene and the primers used were listed in
Finally, the glucose metabolism of S. aureus in biofilms was measured using the Glucose Assay Kit (Beyotime, China), Pyruvate Assay Kit (Solarbio, China), ATP Assay Kit (Beyotime, China), and NAD+/NADH Assay Kit (Beyotime, China), respectively.
10
Metabolic Profile of Leukemia Cells and MDSCs
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Sorted leukemia cells and MDSCs (1 × 105) were lysed, and intracellular ATP, LDH activity, pyruvate content, and NAD+/NADH ratio were measured using the ATP Determination Kit (S0026, Beyotime), the LDH Cytotoxicity Detection Kit (C0017, Beyotime), the Pyruvate Assay Kit (BC2205, Solarbio), and the NAD+/NADH ratio Assay Kit (S0175, Beyotime) respectively, according to the manufacturers' instructions. For Lactate production, sorted leukemia cells and MDSCs (1 × 105) were incubated in StemSpan serum‐free medium (Stem Cell Technologies, 9650) for 18 h at 37 °C. Supernatant cells were then analyzed using the Lactate Assay Kit (BC2235, Solarbio) according to the manufacturer's instructions.
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