The glucose uptake assay (Abcam, Inc.,Cambridge, MA) was performed according to the manufacturer’s instructions. Briefly, pre-adipocytes were differentiated to adipocytes as described and maintained for an additional 4 days. Cultured cells were then starved overnight as described. Cultured cells were washed and starved in KRPH + 2% BSA buffer. Then 1 µM insulin and 2-deoxyglucose (2-DG) were added to activate glucose transporters to take up 2-DG and metabolize it to 2-DG-6-phosphate (2-DG6P). The accumulated 2-DG6P was oxidized to NADPH which generated an oxidized substrate that was measured at OD412nm using a microplate reader. A 2-DG6P standard curve was also measured at the same time. Absorbance values were normalized to the mean “blank” absorbance value. Cells not treated with insulin or 2-DG were used as sample background controls. 2-DG uptake [µM] was calculated as: (Calculated concentration of 2-DG6P/Sample volume) × Sample dilution. The experiment was performed three times, and data were presented as the mean ± s.e.m. Results were analyzed with Microsoft Excel software by applying the equal variance t Test. P ≤ 0.05 was considered statistically significant.
Glucose uptake assay
Manufactured by Abcam
Sourced in United Kingdom, China
The Glucose Uptake Assay is a colorimetric assay kit designed to measure glucose uptake in cells. It provides a simple and sensitive method to quantify glucose levels in cell culture samples, allowing researchers to study cellular glucose metabolism.
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Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Anti phospho gsk3β, by Cell Signaling Technology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti pkm2, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti glut1, by Cell Signaling Technology (1 mentions)
Anti phospho akt substrate, by Cell Signaling Technology (1 mentions)
Myr akt1, by Addgene (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti cyto c, by Cell Signaling Technology (1 mentions)
L lactate assay kit, by Abcam (1 mentions)
Anti ldha, by Cell Signaling Technology (1 mentions)
Anti bak, by Cell Signaling Technology (1 mentions)
Anti bcl xl, by Cell Signaling Technology (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Lactic acid assay kit, by Merck Group (1 mentions)
3 protocols using glucose uptake assay
1
Quantifying Insulin-Stimulated Glucose Uptake
2
Limonin Regulates Glycolysis and Apoptosis in Liver Cancer
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Hep3B and HepG2 cells were obtained from the Cell bank of Chinese Academy of Sciences (Shanghai, People’s Republic of China). By following the instructions of supplier, Hep3B and HepG2 cells were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in a 37°C incubator with 5% CO2. Limonin was purchased from Selleck (Shanghai, People’s Republic of China). The primary antibodies used in Western blotting including anti-Glut1, anti-voltage-dependent anion channel 1 (VDAC-1), anti-LDHA, anti-PKM2, anti-cleaved caspase-3, anti-phospho-Akt substrate, anti-cleaved PARP, anti-Bax, anti-Bak, anti-cyto C, anti-Bcl-2, anti-Bcl-xl, anti-Akt, anti-phospho-Akt, anti-GSK3β, anti-phospho-GSK3β and the secondary HRP-conjugated goat anti-rabbit IgG were products of Cell Signaling Technology (Beverly, MA, USA). The anti-hexokinase-2 antibody, Glucose Uptake Assay and l-Lactate Assay Kits were products of Abcam (Cambridge, UK). The Annexin V-FITC was product of BioLegend (San Diego, CA, USA). For cell transfection, Myr-Akt1 was obtained from Addgene (Cambridge, MA, USA), and the Lipofectamine 3000 was a product of Thermo Fisher Scientific (Waltham, MA, USA).
3
Glucose Uptake Assay for TNBC Cells
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Glucose consumption was determined using a Glucose Uptake Assay (ab136955; Abcam, Shanghai, China). Forty-eight hours after transfection, TNBC cells were subjected to glucose starvation and then incubated with 2-deoxyglucose (2-DG) for 25 min at 37 °C. TNBC cells were washed to remove exogenous 2-DG, lysed, and pipetted. The lysates were heated at 85 °C for 40 min and cooled on ice 5 min. Thereafter, the lysates were neutralized and the supernatant was incubated with reaction mix A for 1 h at 37 °C. Then, the samples were extracted and heated for 40 min at 90 °C followed by cooling on ice for 5 min. Finally, the lysates were incubated with reaction mix B and analyzed using a microplate reader (Bio-Tek). A lactic acid assay kit (Sigma, MO, USA) was used to quantify the lactate levels in TNBC cells.
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