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…

Açıklamalar

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

Etiketler

Play Video

Bu Makaleden Alıntı Yapın
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).

View Video