Following exposure of macrophages to GSK864, citrate was measured using a Citrate Assay Kit (Sigma) as per manufacturer’s instructions.
Citrate assay kit
Manufactured by Merck Group
Sourced in Ireland, United States, Germany
The Citrate Assay Kit is a laboratory equipment product that provides a quantitative method for the determination of citrate concentrations in various sample types. The kit includes all the necessary reagents and components to perform the citrate assay.
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21 protocols using citrate assay kit
1
Macrophage Citrate Assay Protocol
2
Hepatic Lipid and Metabolite Analysis
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Flash frozen liver tissue (50 mg) was homogenized in a TissueLyser II (Qiagen Cat. No./ID: 85300). For TAG and cholesterol assays, liver homogenate was prepared as previously described.[25 ] The dried sample was resuspended in water before TAG and cholesterol analysis. Hepatic TAG (Wako LabAssay Triglyceride kit, Fuggerstraße, Neuss, Germany) and hepatic cholesterol (Wako LabAssay Cholesterol kit) was measured as per manufacturer's instructions. Hepatic citrate (Sigma‐Aldrich Citrate Assay Kit) and lactate (Sigma‐Aldrich Lactate Assay Kit) were measured as per manufacturer's instructions.
3
Glucose, ATP, and Citrate Levels in Cell Lines
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In parallel experiments, both cell lines transfected with shCTRL and shPDK1 were analyzed for glucose levels in the conditioned media and for intracellular ATP concentrations in basal conditions and in response to TKIs. Briefly, conditioned media were removed, centrifuged at 13000× g at 4 °C for 10 min and then assayed for glucose concentrations using the Glucose Assay Kit (GAG020-1KT, Sigma-Aldrich), following manufacturer’s instructions. Intracellular ATP determination was performed using the ATPlite Luminescence Assay (6016941, Perkin Elmer; Waltham, MA, USA) following manufacturer’s instructions. Briefly, cells were lysed, incubated with the ATP reaction mixture for 5 min and then subjected to luminescence measurements. Moreover, citrate levels were determined in shCTRL and shPDK1 cells using the Citrate Assay Kit (Sigma-Aldrich), following manufacturer’s instructions. Absolute glucose, ATP and citrate levels were calculated from the corresponding standard curve and normalized to 106 cells. At least three independent experiments were performed, and data were pooled.
4
Mitochondrial Bioenergetics Profiling
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Cells were transfected and treated with indicated drugs for 24 hours and then collected. Hydroxyl radicals were detected using a Hydroxyl Radical Assay Kit (Jiancheng, Nanjing, China), ATP was measured using an ATP assay kit (Beyotime Biotechnology, Shanghai, China), cell oxygen consumption rate (OCR) was measured using Mito‐Xpress and pH‐Xtra kits (Luxcel Bioscience, Cork, Ireland), mitochondria extraction from cultured cells was performed (MP‐007, Inventbiotech), ACO2 activity was determined using an Aconitase Activity Assay Kit (MAK051, Sigma), and isocitrate and citrate concentrations were determined using an IsoCitrate Assay Kit (MAK319, Sigma) and a Citrate Assay Kit (MAK057, Sigma), respectively. All assays were performed according to the manufacturers' protocols.
5
Citrate Production in HDFs Exposed to SNPs
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Five millilitres of HDFs were cultured in tissue culture polystyrene bottles with a bottom area of 25 cm2. After the cells had reached 90% confluence, the culture media were aspirated, and 2.7 ml of 200 μM SNPs was added. After treatment with SNPs for another 4, 8 or 24 h, the cells were collected by trypsinization, and the samples were prepared according to the instructions of the Citrate Assay Kit (MAK057, Sigma, USA). The citrate content in each sample was measured with a microplate reader. HDFs cultured in culture medium without SNPs were used as controls.
6
Quantifying Extracellular Citrate Levels
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24 hr cultures of BJ1, ST14, their coculture, or blank tubes with citrate, were centrifuged for 10 min at 4000 rpm and the supernatant was sterilized with 0.22 μm filter, prior to deproteination by centrifugation in an Amicon tube (10 kDa). Citrate concentration was measured using a Citrate assay kit (Sigma-Aldrich MAK333).
7
Cytokine Profiling of Cancer Cells
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To measure the release of cytokines, Human Cytokine Antibody Array from RayBiotech was used. Conditioned media were collected from cancer cells as summarised in Fig S3 . Cytokine values from the unconditioned media used for conditioning were subtracted from the values obtained in the media from cancer cells. Consequently, cytokine levels in the media from cancer cells were subtracted from the values obtained in the media from fibroblasts. The kit was used according to the manufacturer’s instructions and analysed using supplied positive and negative controls for all the assays. We used the same control media for all conditions tested, and all the membranes were prepared at the same time. The values between different cell types were not compared, but the trend in cytokines released is presented on separate graphs for all conditions tested.
Citrate in the media from fibroblasts was measured using Citrate assay kit (Sigma-Aldrich) according to the manufacturer instructions.
Citrate in the media from fibroblasts was measured using Citrate assay kit (Sigma-Aldrich) according to the manufacturer instructions.
8
Citrate Assay Protocol for Bacterial Cultures
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Citrate levels were measured using the citrate assay kit (MAK057) from Sigma-Aldrich. Strains were grown overnight in BM, diluted into fresh BM to an OD of 0.1, and grown for 5 h at 37°C with continuous shaking. For each strain, 1 × 108 cells in 100 μL citrate assay buffer were homogenized in a 1.5-mL microcentrifuge tube containing 100 μL glass beads with a FastPrep instrument at 6.5 m/s for 60 s. After centrifugation for 10 min at maximum speed, 30 μL of supernatant was pipetted into a 96-well plate. Twenty microliters of citrate assay buffer was added to reach the final volume of 50 μL described in the kit manual. Reaction mixes were prepared, and the analysis was carried out as described in the manufacturer’s manual.
9
Bacterial Cultivation and Citrate Utilization
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All bacterial strains (listed in Table S1 in the supplemental material) were cultured aerobically at 37°C unless stated otherwise in the particular section. Cells were cultured in lysogenic broth (LB) or on agar plates (LA) with the appropriate amount of the following antibiotic(s) or inducer when needed: carbenicillin, 100 μg/mL; tetracycline, 15 μg/mL; and 1 mM IPTG (isopropyl-β-d -thiogalactopyranoside; Sigma). To test for the ability to use citrate, we used modified Simmons’s medium, as previously described in reference 57 (link). One representative of three independent experiments is shown. Incubation in Koser’s citrate broth (Condalab) was done overnight, and where necessary, 0.1% glucose, 15 mM nitrate, and 15 mM fumarate were included. The citrate levels were measured with a citrate assay kit (Sigma-Aldrich), following the manufacturer’s instructions.
10
Bacterial Citrate Assay Protocol
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Bacterial strains were diluted from overnight culture into fresh LSM at an initial OD of approximately 0.1 and cultured to mid-log phase (OD600 = 1.0) in LSM (250-ml flask without baffles, 25 ml of medium, 220-rpm shaking, 37°C), and harvested bacteria were washed in PBS, resuspended in an equal volume of citrate assay buffer (see below), and immediately frozen in liquid nitrogen. Bacteria were then thawed and lysed by bead-beating using 0.1-mm zirconia-silica beads (BioSpec) for 15 min. Citrate concentrations were determined using the citrate assay kit (Sigma) according to the manufacturer’s instructions.
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