Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo
Published: April 30, 2023
Abstract
Source: Kumar, S. B. et al., Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo. J. Vis. Exp. (2022)
In this video, we demonstrate an ex vivo technique to measure extracellular glucose analog depletion in harvested mouse organs. It involves incubating the mouse tissue in a solution containing fluorescently-labeled deoxyglucose, which is taken up by the cells. The glucose analog uptake can be indirectly determined by measuring the fluorescence of the incubated medium.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. NOTE: All procedures must be done in a class II biosafety cabinet with the blower on and the lights off. 1. Preparation of materials NOTE: All materials are listed in the Table of Materials. Prepare Medium 1, centrifugation Mediu…
Representative Results
Figure 1: Kinetics of extracellular FD-glucose depletion in different organs ex vivo. (A-C) Lepob mice were injected with vehicle (PBS, n = 4), insulin (12 IU/kg BW, n = 3), and AAC2 (1 nmol/g BW, n = 3). After 15 min, tissues were dissected and isolated. Explants of (A) visceral fat, (B) liver, an…
Divulgazioni
The authors have nothing to disclose.
Materials
| 96-well plates | Falcon | 353227 | Plastic ware |
| B6.V-Lepob/J male mice | Jackson Laboratory | stock number 000632 | Mice |
| BioTek Synergy H1 modular multimode microplate reader (Fisher Scientific, US) | Fisher Scientific, US | Fisher Scientific, US | Device |
| Cell incubator | Forma | Series II Water Jacket | Device |
| Diet (mouse/rat diet, irradiated) | Envigo | Teklad LM-485 | Diet |
| Isoflurane, 5% | Henry Schein | NDC 11695-6776-2 | Anestaetic |
| Phosphate buffered solution | Sigma-Aldrich | DA537-500 mL | Cell culture |
| Penicillin/streptomycin (P/S) | Gibco/ThermoFisher | 15140-122 | Cell culture |
| Glucose-free and phenol red-free DMEM | Gibco/ThermoFisher | A14430-01 | Cell culture |
| Fluorescent 2-deoxy-2-[(7-nitro-2,1,3- benzoxadiazol-4-yl) amino]-Dglucose) | Sigma | 72987-1MG | Assay |
| Dulbecco's Modified Eagle Medium | Gibco/ThermoFisher | 11965-092 | Cell culture |
| Bovine serum | Gibco/ThermoFisher | 161790-060 | Cell culture |
| Ethanol | Sigma-Aldrich | E7023-500mL | Reagent |
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Citazione di questo articolo
Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo. J. Vis. Exp. (Pending Publication), e21213, doi: (2023).