Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo

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.

1. Prepare Medium 1, centrifugation Medium 2, and storage and working solutions of fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG or FD-glucose) according to

Results

Figure 1: Kinetics of extracellular FD-glucose depletion in different organs ex vivo. (A-C) Lep^ob mice were injected with vehicle (PBS, n = 4), insulin (12 IU/kg BW, n = 3), and AAC2 (1 nmol/g BW, n = 3). After.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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