Secreted L-lactate concentrations in cell-free culture supernatants were determined by using the Glycolysis Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, Michigan, USA). For intracellular L-lactate determination, cryopreserved cells were allowed to recover for 24 h in supplemented RPMI-1640 medium and suspensions were stained using the Glycolysis Cell-Based Assay Kit (Cayman Chemical). The cells were washed once in wash buffer (0.5% FCS/1× PBS), stained with cell surface markers, and resuspended in 1× PBS prior to analysis. The highly colored intracellular formazan was detected in the FL3 channel on a FACSCalibur.
Glycolysis cell based assay kit
Manufactured by Cayman Chemical
Sourced in United States
The Glycolysis Cell-Based Assay Kit is a laboratory tool used to measure the rate of glycolysis, a metabolic process that converts glucose into energy, in living cells. The kit provides the necessary reagents and protocols to quantify the production of lactate, a byproduct of glycolysis, in a microplate format.
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Lab products found in correlation
Glucose uptake cell based assay kit, by Cayman Chemical (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Atp bioluminescence assay kit cls 2, by Roche (2 mentions)
Synergy ht microplate reader, by Agilent Technologies (2 mentions)
Glucose colorimetric assay kit, by Cayman Chemical (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Celltiter glo 2.0 assay kit, by Promega (1 mentions)
Filtermax f5 plate reader, by Molecular Devices (1 mentions)
Infinite f200, by Tecan (1 mentions)
2 deoxy d glucose 2 dg, by Thermo Fisher Scientific (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
Glucose uptake colorimetric assay kit, by Abcam (1 mentions)
Glutamine colorimetric assay kit, by Abcam (1 mentions)
Atp detection assay kit, by Cayman Chemical (1 mentions)
Ldh activity colorimetric assay kit, by Abcam (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Spectramax m5 spectrophotometer, by Molecular Devices (1 mentions)
Glucose assay kit, by Merck Group (1 mentions)
80 protocols using glycolysis cell based assay kit
1
Quantifying Intracellular Lactate Levels
2
Glucose Uptake and Lactate Production
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The glucose absorption was analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) by using Glucose Uptake Cell-Based Assay Kit (No.600470, Cayman Chemical). Lactate production was determined using Glycolysis Cell-Based Assay Kit (No.600450, Cayman Chemical) according to the manufacture’s protocol.
3
Glycolysis and Intracellular ATP Assays
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Glycolysis and intracellular ATP levels were measured using the Glycolysis Cell-Based Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) and the CellTiter-Glo 2.0 Assay Kit (Promega, Madison, WI, USA), respectively, following the manufacturers’ instructions.
4
Quantifying Cellular Glycolysis Levels
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Cells were plated in RPMI-1640 containing 8% FBS. Supernatants were collected from treated/infected cells at 18–24 h post-infection. L -lactate, the end product of glycolysis was measured using the Glycolysis Cell-Based Assay kit from Cayman Chemical (Michigan, USA), according to manufacturer's instructions. Absorbance was then measured at 450 nm using a FilterMax F5 plate reader (Molecular Devices).
5
Glycolysis Assay for Erythroid Cells
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Example 62
The erythroid cell population may be verified as metabolically active using a variety of different enzyme based assays to quantify important metabolic end products. Active glycolysis is a crucial metabolic pathway to assess and may be measured with the following assay (Glycolysis cell-based assay kit, Cayman Chemical, Item 600450).
450 ul of assay buffer is aliquoted into a test tube, followed by 50 uL of the L-Lactic acid standard asnd mixed thoroughly. A titration curve is constructed using the lactic acid concentration standard, beginning with a 1 mM dilution.
Cells are added to a 96 well plate and centrifuged at 1000 RPM for 5 minutes. 100 uL of the standards are transferred into a separate 96 well plate. 90 uL of assay buffer is then added to each well. 10 ul of supernatant in each cell well is then transferred to corresponding new wells. Add 100 ul of reaction solution to each well using a repeating pipettor. The plates are then incubated on an orbital shaker for 30 minutes at RT. The absorbance isread at 490 nm with a plate reader. Results are compared to natural cells to identify any metabolic differences.
6
Glycolysis Profiling of Breast Cancer Stem Cells
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The glycolysis rate of breast cancer stem cells was revealed by measuring the levels of L-lactate, using the Glycolysis Cell-Based Assay Kit (Cayman, Ann Arbor, Michigan, USA). The assay was performed according to the manufacturer’ s protocol. Briefly, cells were cultured in a 96-well plate, and the following day were treated with GW6471 for 72 h while the control cells received only the culture medium. After 72 h, the culture supernatant was removed from each well and added to the reaction solution. The mixture was incubated with gentle shaking on an orbital shaker for 30 min at room temperature, and the absorbance at 490 nm was detected with a microplate reader Infinite F200 (Tecan, Morrisville, NC). Data were expressed as mM.
7
Quantification of Extracellular L-Lactate
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Analysis was conducted by a kit (Glycolysis Cell Based Assay Kit Cayman Chemical Company, Ann Arbor, MI, USA) for the quantification of extracellular L-Lac levels (the end product of glycolysis) which are proportionally correlated with intra-cellular glycolytic activity. Briefly, cells deriving from 2 different cultures for each NSC type (15.000 cells/well) were plated in duplicate in 96 wells and cultured for 60 hours. Afterwards, standards and samples (10 µl of cell conditioned media as well as an equal amount of cell-free media, NSC and OEC medium) were processed and analyzed following the manufacturer’s instructions. L-Lac levels were calculated subtracting the absorbance levels of the corresponding cell-free media and corrected for the total cell number in each well.
8
Assessing Mitochondrial Activity and Glycolysis
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Cells sorted by TMRM fluorescence were collected and assessed for ATP production using the ATP Bioluminescence Assay Kit CLS II (Roche). 30,000 cells were collected per biological replicate (n = 3) and each was assayed in technical triplicate so that each assay well had 7,500 cells. L-lactate production was assayed against an L-Lactate standard curve by a Glycolysis Cell-Based Assay Kit (Cayman Chemicals) and results were normalized to cell number. 50 μM 2-deoxy-D-glucose (2-DG, Acros Organics) was added to control cells 24 h prior to analysis. Plates were read on a Synergy H1 plate reader (BioTek).
9
Myogenic Lactate Accumulation Assay
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Following 21 days of myogenic induction, spent media were replaced with fresh media. Cells were incubated 2 hours at 37°C, and then l -lactate accumulation was measured in the media using the Glycolysis Cell-Based Assay Kit (Cayman Chemical). Data were corrected for values in a no-cell control well and for total protein content, as measured by BCA method.
10
Glycolysis Dynamics in CD4+ T cells
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CD4+ T cells were cocultured with SKOV3 for 5 days or treated with ssRNA40 or ssRNA41 for 24 h. Glucose uptake was measured by Glucose Uptake Colorimetric Assay Kit (Biovision). L-lactate concentrations in cell culture supernatants from CD4+ T cells were determined using the Glycolysis Cell-Based Assay Kit (Cayman). The CD4+ T cells and culture supernatant was collected and glucose uptake and L-lactate concentrations determined according to the manufacturer’s protocols.
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