Metabolism Assays
Glucose Uptake Assay Kit (Fluorometric) (BA0102)
- SKU:
- BA0102
- Product Type:
- Assay
- Detection Method:
- Fluorometric
- Instrument:
- Microplate Reader
- Sample Type:
- Cell Culture
- Research Area:
- Glucose Uptake
- Research Area:
- Diabetes & Obesity
Description
Glucose Uptake Assay Kit (Fluorometric) (BA0102) (BA0102)
The Glucose Uptake Assay Kit (Fluorometric) (SKU: BA0102) is a fluorescent, cell-based assay for measuring glucose uptake in whole cells. It uses 2-deoxyglucose (2-DG), a glucose analogue taken up by glucose transporters and phosphorylated by hexokinase to 2-deoxyglucose 6-phosphate (2-DG6P), which accumulates intracellularly. After lysis, excess NADP and glucose 6-phosphate dehydrogenase metabolise 2-DG6P to generate NADPH, which is measured through a recycling reaction that amplifies the signal. The resulting fluorescence at 530/585 nm is proportional to the 2-DG6P concentration. The kit is non-radioactive and can be automated as a medium-throughput assay.
| Product Name: | Glucose Uptake Assay Kit (Fluorometric) (BA0102) |
| SKU: | BA0102 |
| Detection Method: | Fluorometric cell-based (530/585 nm) |
| Detection Range: | 0.1 - 5 uM 2-DG6P |
| Sample Type: | Whole cells (adherent or suspension) |
| Species Reactivity: | All |
| Assay Time: | Multi-step protocol (overnight culture plus enzymatic detection) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A non-radioactive, cell-based glucose uptake assay that quantifies 2-DG6P accumulation via an amplified NADPH recycling reaction. Suitable for evaluating the effect of ligands and drugs on glucose transport.
- Safe, no radioactive material used
- Sensitive and accurate with a detection limit of 0.1 uM and linearity up to 5 uM 2-DG6P
- Simple, convenient and automatable as a medium-throughput assay
- Determination of glucose uptake in whole cells
- Evaluation of the effects of ligands or drugs on glucose transport
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Culture, starve and treat cells: seed cells into a 96-well culture plate and incubate. Incubate with low-glucose, low-serum media to raise glucose demand, wash twice with 37C PBS, then starve in glucose-free, serum-free media for 40 minutes, adding any treatments at this step. |
| 2 | Add 2-deoxyglucose: add 10 uL of 2-DG substrate per well and incubate for 20 minutes. Remove media and wash three times with ice-cold PBS. |
| 3 | Lyse and extract 2-DG6P: add Lysis Buffer (1% Triton X-100 in NADP Extraction Buffer), shake, then heat at 80C for 10 minutes. Prepare the 2-DG6P standard curve, add NADPH Extraction Buffer, cool the plate, then add Working Reagent 1 (Assay Buffer, Enzyme A, NADP) and incubate at 37C for 60 minutes. |
| 4 | Extract and measure NADPH: add NADPH Extraction Buffer and heat, add NADP Extraction Buffer and cool, then transfer 50 uL of each sample and standard to a black 96-well plate. Prepare Working Reagent 2 with diluted Enzyme A, Enzyme B, G6P Reagent and Probe. |
| 5 | Add 50 uL Working Reagent 2 per well and read at 530/585 nm over 20 minutes, recording fluorescence at time zero (F0) and at 20 minutes (F20). |
Compute delta-F for each standard and sample by subtracting F0 from F20. Plot the standard delta-F values and determine the slope. [2-DG6P] = (delta-F_Sample - delta-F_Blank) / Slope x n, where delta-F_Blank is the value of the water standard and n is the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| G6P Reagent | 1.5 mL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| 2-DG Substrate | 1.2 mL | -20C |
| NADP | 120 uL | -20C |
| 2-DG6P Standard | 120 uL | -20C |
| Probe | 750 uL | -20C |
| NADP Extraction Buffer | 12 mL | -20C |
| NADPH Extraction Buffer | 12 mL | -20C |
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