Name: Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina (Video) | JoVE Journal
Uploaded: 2025-03-14T15:00:00.000+00:00
Duration: 7 min 4 s
Description: Learn to measure glucose uptake in intact murine neural retina using the fluorescent analog 6-NBDG. This ex vivo protocol enables rapid, cost-effective assessment of retinal metabolism for aging and disease research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was funded by unrestricted departmental funds awarded to Lauren K. Wareham.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Vanderbilt University Medical Center.
1. Preparation for assay
NOTE: Preparation should be carried out on the day of the assay immediately prior to running the assay. This is necessary due to the time-sensitive nature of the protocol.
<ol>
	<li>General preparation ahead of the experiment 
	NOTE: 6-NBDG is light-sensitive and should be protected from light. Cover all 6-NBDG solutions with aluminum foil.
	<ol>
		<li>A stock solution of 5 mM 6-NBDG in 50% DMSO/1x PBS is stable at -20 &#176;C for 6 months. Make a stock solution and store 500 &#181;L aliquots for future assays.</li>
		<li>Place a 50 mL conical tube of Neurobasal-A media (without glucose) on ice.</li>
		<li>Make sure that the water bath is set to 37 &#176;C.</li>
		<li>Label 4&#160;&#215; 1.5 mL microcentrifuge tubes per retina: 1) glucose starvation, 2) 6-NBDG incubation, 3) sonication, and 4) collection tube for sample for Pierce assay (i.e., if you have four retina in total, you will need 16 microcentrifuge tubes). See Figure 1 for a summary of the tube setup.</li>
		<li>Add 200 &#181;L of neurobasal A medium from the 50 mL conical tube on ice to the pre-labeled sonication tubes and set on ice for step 2.5.4.</li>
		<li>Power on the plate reader.</li>
		<li>Set up plate information in plate reader software using the fluorescence endpoint assay feature with the following parameters: excitation 483 nm, emission 550 nm, and cut-off 530 nm (parameters predetermined for 6-NBDG).</li>
	</ol>
	</li>
	<li>Reagent preparation ahead of the experiment: 6-NBDG
	<ol>
		<li>Make a working solution of 6-NBDG in neurobasal-A media at a final concentration of 500 &#181;M by diluting the 5 mM stock made in step 1.1.1. 
		NOTE: Remember to cover all 6-NBDG solutions with aluminum foil. Make an appropriate volume of 500 &#181;M 6-NBDG working solution for the entire assay (need 200 &#181;L of working 6-NBDG solution per retina; include extra volume for error and amounts needed for creating standard solutions in step 2.4.).</li>
		<li>Pipette 200 &#181;L of 6-NBDG working solution into the pre-labeled tubes made in step 1.1.4 for the 6-NBDG incubation step.</li>
	</ol>
	</li>
</ol>
2. Running the 6-NBDG uptake assay
NOTE: See Figure 2 for a step-by-step overview.
<ol>
	<li>Retinal tissue dissection
	<ol>
		<li>Sedate the mouse using inhaled isoflurane anesthesia (2% isoflurane in 5% carbon dioxide/95% oxygen). 
		NOTE: Confirm that the mouse is adequately anesthetized by pinching the foot pad on both hind feet. If properly anesthetized, there should be no jerking motion (pedal withdrawal reflex.) If there is a reflex movement, return to step 2.1.1.</li>
		<li>Euthanize the mouse by cervical dislocation followed by decapitation and then rapidly enucleate the eyes. Place the enucleated eyes into a Petri dish filled with 5-10 mL of ice-cold neurobasal-A media (without glucose) on ice. 
		NOTE: Below are basic guidelines for neural retinal dissection (excluding retinal pigment epithelium and choroid); for more in-depth instruction and guidance, refer to a previously published protocol11 and follow the section &#34;mouse retinal dissection&#34;.</li>
		<li>Place the Petri dish under a dissection microscope on ice.</li>
		<li>Fold a lint-free wipe into a small square and dampen it with Neurobasal A media.</li>
		<li>Transfer an eye onto the damp, lint-free wipe using a plastic transfer pipette with the end trimmed off. 
		NOTE: The lint-free wipe provides increased friction to keep the eye from moving during dissection.</li>
		<li>Take a 26 G needle and puncture the side of the cornea anterior to the limbus.</li>
		<li>Use Vannas scissors to cut along the limbus from the point of puncture to remove the cornea.</li>
		<li>Once the cornea is removed, grasp the sclera midway between the limbus and the optic nerve head with a pair of forceps. Using another pair of forceps in the opposite hand, grasp the lens and remove it quickly in one swift motion. 
		NOTE: If the lens is removed slowly, the retina will stay attached to the lens and become difficult to dissect.</li>
		<li>Transfer the eye cup to a Petri dish with cold Neurobasal A on ice.</li>
		<li>Using forceps in both hands, take the eye cup and carefully hold the cup at the ora serrata.
		<ol>
			<li>To release the retinal cup from the sclera, clamp down on the sclera using forceps, taking care not to clamp the retinal tissue.</li>
			<li>Slide the forceps carefully between the scleral tissue and retinal tissue around the entire globe until the entire retina is released from the limbus.</li>
		</ol>
		</li>
		<li>Using Vannas scissors, make a small incision in the sclera at the limbus.</li>
		<li>Take forceps to grab the edges of the incision and peel apart the scleral tissue down to the optic nerve head region. Grasp the sclera using forceps in the non-dominant hand and gently guide the retina away from the sclera using closed forceps in the dominant hand.</li>
		<li>Snip the optic nerve head with scissors to detach the retina completely from the sclera and any optic nerve that remains.</li>
		<li>Transfer the retina using a trimmed transfer pipette into the pre-prepared 1.5 mL microcentrifuge tube for glucose starvation from step 1.1.4, which contains 200 &#181;L of neurobasal-A media (without glucose)-1 retina per tube.</li>
	</ol>
	</li>
	<li>Glucose starvation step
	<ol>
		<li>Transfer the tubes from step 2.1.14 into a 37 &#176;C water bath and incubate at 37 &#176;C for 20 min.</li>
	</ol>
	</li>
	<li>NBDG incubation
	<ol>
		<li>Transfer each retina to a new pre-labeled tube for 6-NBDG incubation from step 1.1.4, which contains 200 &#181;L of 500 &#181;M 6-NBDG in neurobasal-A medium from step 1.2.2. 
		NOTE: The retina should be transferred using a transfer pipette that has been cut to enlarge the tip so as to transfer the retina with minimal damage.&#160;Additionally, this can be done with tweezers if confident in one&#8217;s ability to transfer retina without damage.</li>
		<li>Transfer tubes to 37 &#176;C water bath and incubate at 37 &#176;C for 60 min.</li>
	</ol>
	</li>
	<li>Preparation of 6-NBDG standards (concentration range 0 &#181;M-40 &#181;M) 
	&#8203;NOTE: Prepare the standards during the 60 min 6-NBDG incubation period (step 2.3.2.). Standards can be prepared at room temperature. Protect solutions from light.
	<ol>
		<li>Label 1.5 mL microcentrifuge tubes #1-8 for the 8 standards (see Figure 3).</li>
		<li>For standard #1 (40 &#181;M 6-NBDG), pipette 32 &#181;L of 500 &#181;M 6-NBDG stock solution made in step 1.2.1.</li>
		<li>Add 368 &#181;L of neurobasal-A media into the microcentrifuge tube and mix thoroughly by pipetting up and down.</li>
		<li>For standards #2-8, serially dilute samples as follows:
		<ol>
			<li>Pipette 200 &#181;L of neurobasal-A media into tubes #2-8.</li>
			<li>Take 200 &#181;L from standard tube #1 and pipette it into tube #2, then mix thoroughly with the pipette by pipetting up and down.</li>
			<li>Take 200 &#181;L from standard tube #2 and pipette into standard tube #3. Mix thoroughly with the pipette by pipetting up and down.</li>
			<li>Take 200 &#181;L from standard tube #3 and pipette it into tube #4, then mix thoroughly by pipetting it up and down.</li>
			<li>Take 200 &#181;L from standard tube #4 and pipette it into tube #5, then mix thoroughly by pipetting it up and down.</li>
			<li>Take 200 &#181;L from standard tube #5 and pipette it into tube #6, then mix it thoroughly by pipetting it up and down.</li>
			<li>Take 200 &#181;L from standard tube #6 and pipette it into tube #7, then mix it thoroughly by pipetting it up and down.</li>
			<li>Add no additional solution to tube #8 (i.e., it should contain only 200 &#181;L of neurobasal-A media added in step 2.4.4.1.).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Washing, sonication, and centrifugation for sample collection
	<ol>
		<li>Remove all tubes from the 37 &#176;C water bath once the 60-min 6-NBDG incubation is complete.</li>
		<li>Wash each retina with 500 &#181;L of ice-cold neurobasal-A media as quickly and carefully as possible.
		<ol>
			<li>To wash, remove the 6-NBDG solution with a pipette, making sure not to touch or disturb the retinal tissue. 
			NOTE: There is also the option to transfer the retina with tweezers from the 6-NBDG incubation tube to a fresh tube that has 500 uL of neurobasal-A media. Then, you only have to do 3 additional washes. Do this if you are comfortable with your ability to not damage the retina with tweezers.</li>
			<li>Then, discard the solution in an appropriate waste receptacle. Immediately pipette 500 &#181;L of cold neurobasal- A medium into the same tube to replace the solution that was removed. Replace tubes on ice in between washes.</li>
		</ol>
		</li>
		<li>Repeat 3 more times for a total of 4 washes.</li>
		<li>Using a transfer pipette with trimmed edge or tweezers, carefully transfer the retina to the sonication tubes containing neurobasal A medium on ice (see step 1.1.4).</li>
		<li>Chop the retina into small pieces using small dissection scissors. 
		NOTE: Make sure to wash tools in ethanol between chopping each retina so as not to cross-contaminate other samples.</li>
		<li>Sonicate each retina for 5-10 s on an amplitude of 10 and immediately place it back on ice. 
		NOTE: Make sure to clean the sonication probe between samples with 100% ethanol followed by ultrapure H2O. At this point, either pre-cool the centrifuge to 4 &#176;C or utilize the centrifuge in a 4 &#176;C walk-in cold room if available.</li>
		<li>Centrifuge at 4 &#176;C for 15 min at 21130 x&#160;g or maximum speed if less than 21130 x g. 
		NOTE: During the 15-min centrifuge in step 2.5.7, pipette 100 &#181;L of standards #1-8 made in step 2.4 into a black, clear bottom 96-well plate.</li>
	</ol>
	</li>
	<li>Reading samples on a plate reader
	<ol>
		<li>After the 15-min centrifuge is over, remove 100 &#181;L of supernatant and pipette into the 96-well plate.</li>
		<li>Insert plate into plate reader. 
		NOTE: Plate should have 100 &#181;L of each sample and 100 &#181;L of each standard.</li>
		<li>In the plate reader software, select fluorescence endpoint assay with parameters selected in step 1.1.7.</li>
		<li>Run the assay and save the results.</li>
		<li>Pipette samples from 96-well plate into the pre-labeled tubes from 1.1.4 for the Pierce assay. 
		&#8203;NOTE: At this point, samples may be immediately assayed for protein (see below) or stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Pierce assay for protein normalization 
	&#8203;NOTE: The Pierce assay should be run in duplicate (both standards and samples) for increased accuracy.
	<ol>
		<li>Using the Pierce assay pre-diluted protein assay standards, pipette 10 &#181;L of each in duplicate into a 96-well clear bottom plate.</li>
		<li>Pipette 10 &#181;L of each of the samples in duplicate from step 2.6.5 into the 96-well plate.</li>
		<li>Add 150 &#181;L of Pierce reagent to all wells containing standards or samples. Cover the 96-well plate and mix on a plate shaker at medium speed for 1 min.</li>
		<li>Incubate the plate at room temperature (RT) for 5 min.</li>
		<li>Insert plate into plate reader. Select absorbance end point assay and set the wavelength to 660 nm.</li>
		<li>Measure the absorbance of standards and samples and save the results. Calculate the total microgram protein in each sample by plotting a standard curve.</li>
	</ol>
	</li>
	<li>Calculating retinal 6-NBDG uptake. 
	NOTE: 6-NBDG results need to be normalized to total protein content due to uneven sizes of the retina.
	<ol>
		<li>Using the fluorescence intensity values obtained in step 2.6, use the fluorescence readings from predetermined standards to plot a 6-NBDG fluorescence standard curve and quantify 6-NBDG concentration (&#181;M) in each sample.</li>
		<li>Normalize sample results to corresponding protein amounts to determine [6-NBDG] &#181;M/&#181;g protein.</li>
	</ol>
	</li>
</ol>

<ol><li>Casson, R. J., Chidlow, G., Crowston, J. G., Williams, P. A., Wood, J. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+energy+metabolism+in+health+and+glaucoma%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Retinal energy metabolism in health and glaucoma.</a> Prog Retin Eye Res. 81, 100881(2021).</li>
<li>Daniele, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+uptake+by+GLUT1+in+photoreceptors+is+essential+for+outer+segment+renewal+and+rod+photoreceptor+survival%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Glucose uptake by GLUT1 in photoreceptors is essential for outer segment renewal and rod photoreceptor survival.</a> FASEB J. 36 (8), e22428(2022).</li>
<li>Fernandes, R., Hosoya, K. -I., Pereira, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactive+oxygen+species+downregulate+glucose+transport+system+in+retinal+endothelial+cells%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Reactive oxygen species downregulate glucose transport system in retinal endothelial cells.</a> Am J Physiol Cell Physiol. 300 (4), C927-C936 (2011).</li>
<li>Badr, G. A., Tang, J., Ismail-Beigi, F., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+downregulates+GLUT1+expression+in+the+retina+and+its+microvessels+but+not+in+the+cerebral+cortex+or+its+microvessels%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Diabetes downregulates GLUT1 expression in the retina and its microvessels but not in the cerebral cortex or its microvessels.</a> Diabetes. 49 (6), 1016-1021 (2000).</li>
<li>Kumagai, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+transport+in+brain+and+retina%3A+implications+in+the+management+and+complications+of+diabetes%5BTitle%5D)+AND+%22Diabetes+Metab+Res+Rev%22%5BJournal%5D)">Glucose transport in brain and retina: implications in the management and complications of diabetes.</a> Diabetes Metab Res Rev. 15 (4), 261-273 (1999).</li>
<li>Winkler, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GLUT1+reductions+exacerbate+Alzheimer%27s+disease+vasculo-neuronal+dysfunction+and+degeneration%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">GLUT1 reductions exacerbate Alzheimer's disease vasculo-neuronal dysfunction and degeneration.</a> Nat Neurosci. 18 (4), 521-530 (2015).</li>
<li>Mosconi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypometabolism+exceeds+atrophy+in+presymptomatic+early-onset+familial+Alzheimer%27s+disease%5BTitle%5D)+AND+%22J+Nucl+Med%22%5BJournal%5D)">Hypometabolism exceeds atrophy in presymptomatic early-onset familial Alzheimer's disease.</a> J Nucl Med. 47 (11), 1778-1786 (2006).</li>
<li>Moreno, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+and+morphometric+changes+in+rat+optic+nerve+microvessels+in+a+glaucoma+experimental+model%5BTitle%5D)+AND+%22Arch+Soc+Esp+Oftalmol%22%5BJournal%5D)">Morphological and morphometric changes in rat optic nerve microvessels in a glaucoma experimental model.</a> Arch Soc Esp Oftalmol. 89 (12), 471-476 (2014).</li>
<li>Barros, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+validation+of+6-NBDG+as+a+probe+for+the+glucose+transporter+GLUT1+in+astrocytes%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Kinetic validation of 6-NBDG as a probe for the glucose transporter GLUT1 in astrocytes.</a> J Neurochem. 109 (s1), 94-100 (2009).</li>
<li>Hamilton, K. E., Bouwer, M. F., Louters, L. L., Looyenga, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+binding+and+uptake+of+fluorescent+glucose+analogs+2-NBDG+and+6-NBDG+occurs+independent+of+membrane+glucose+transporters%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters.</a> Biochimie. 190, 1-11 (2021).</li>
<li>Feigenspan, A., Babai, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+horizontal+slices+of+adult+mouse+retina+for+electrophysiological+studies%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation of horizontal slices of adult mouse retina for electrophysiological studies.</a> J Vis Exp. 119, e55173(2017).</li>
<li>Lundgaard, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+neuronal+glucose+uptake+heralds+activity-dependent+increases+in+cerebral+metabolism%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism.</a> Nat Commun. 6 (1), 6807(2015).</li>
<li>Zou, C., Wang, Y., Shen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2-NBDG+as+a+fluorescent+indicator+for+direct+glucose+uptake+measurement%5BTitle%5D)+AND+%22J+Biochem+Biophys+Methods%22%5BJournal%5D)">2-NBDG as a fluorescent indicator for direct glucose uptake measurement.</a> J Biochem Biophys Methods. 64 (3), 207-215 (2005).</li>
<li>Wareham, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solving+neurodegeneration%3A+common+mechanisms+and+strategies+for+new+treatments%5BTitle%5D)+AND+%22Mol+Neurodegener%22%5BJournal%5D)">Solving neurodegeneration: common mechanisms and strategies for new treatments.</a> Mol Neurodegener. 17 (1), 23(2022).</li>
</ol>

The authors have nothing to disclose.

In summary, the method described enables basic science researchers to measure uptake of the fluorescent glucose analog, 6-NBDG, in ex vivo murine neural retina. Glucose is an essential metabolite for the neural retina, it's uptake supports the high rates of glycolysis and mitochondrial respiration needed to produce energy in the form of adenosine triphosphate (ATP)1. Since glucose is the preferred energy substrate, many retinal cells express glucose transporters (GLUTs) to facilitate the ...

Glucose is an essential metabolite for the neural retina, where it is used to fuel high rates of glycolysis and mitochondrial respiration to produce energy in the form of adenosine triphosphate (ATP)1. Since glucose is the preferred energy substrate, many retinal cells express glucose transporters (GLUTs) to facilitate the uptake of glucose from the vasculature and surrounding tissue2. GLUTs comprise a family of intrinsic membrane glycoproteins that are responsible for glucose transport into mammalian cells3. GLUT transporter-1 (GLUT1) is the primary glucose transporter in the retina, expressed throughout the retinal layers4 and by capillary endothelial cells that comprise the blood-retinal barrier (BRB)5. Interestingly, in neurodegenerative diseases of the central nervous system (CNS), including Alzheimer's disease, a reduction in GLUT1 protein levels and glucose uptake precede brain atrophy and neuronal dysfunction in humans6,7. In a rat model of ocular hypertension, lower levels of GLUT1 were also observed in capillaries8. Reduced transport of glucose into the outer retina is implicated in photoreceptor loss in animal models of human retinitis pigmentosa and may also play a role in retinal neurodegeneration, such as that observed in glaucoma. Therefore, an understanding of glucose transport in the neural retina is required to establish its role in retinal neurodegeneration.
Here, we describe a novel, inexpensive, and straightforward biochemical method for measuring 6-NBDG uptake in ex vivo murine neural retina, i.e., excluding retinal pigmented epithelium and choroid. Compared with other fluorescent analogs such as 2-NBDG, 6-NBDG is composed of a glucose moiety on which a fluorescent nitrobenzoxydiazoamino group replaces the hydroxyl group at carbon 6, preventing phosphorylation by hexokinase and further metabolic breakdown9. Although 6-NBDG has high specificity for GLUT1, with a binding affinity 300 times higher than glucose9, we detect uptake of this analog in the presence of GLUT1 inhibitors10. As such, this assay provides a relatively quick and inexpensive method to measure glucose uptake ex vivo in the whole mouse retina, which is partially GLUT1 mediated. 
Measurement of glucose uptake in tissue in real-time is challenging, often requiring radioisotope labeling or high-resolution imaging methods. Here, we employ a fluorescent biochemical assay to quickly determine 6-NBDG uptake across multiple retinal samples in ex vivo conditions. The protocol provides information about total retinal glucose uptake; it does not provide information about retinal cell-specific levels of 6-NBDG uptake.

<table><tbody><tr><td># 5 forceps</td><td>Katena</td><td>K5-6550</td><td>Used for retina dissection</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Thermo Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>26 G x 5/8&quot; needle</td><td>sol-M</td><td>112658</td><td>Used to puncture cornea during dissection</td></tr><tr><td>5 mL tubes</td><td>MTC bio</td><td>c2540</td><td></td></tr><tr><td>50 mL tubes</td><td>Avantor by VWR</td><td>89039-656</td><td></td></tr><tr><td>6-NBDG</td><td>Invitrogen</td><td>N23106</td><td>Fluorescent gucose analog</td></tr><tr><td>96 well plates black with clear bottom</td><td>Thermo Fisher Scientific</td><td>265301</td><td></td></tr><tr><td>Anesthetic Charcoal Filter Cannister</td><td>ReFresh</td><td>EZ-258</td><td>Used in anesthesia set up</td></tr><tr><td>BAY-876</td><td>Millipore Sigma</td><td>SML1774</td><td>For inhibition of GLUT1.</td></tr><tr><td>Centrifuge at 4 &amp;deg;C</td><td>Eppendorf</td><td>EPP-5424</td><td></td></tr><tr><td>Compressed gas (5% carbon dioxide, 95% oxygen)</td><td>Airgas</td><td>UN3156</td><td>Used in anesthesia set up</td></tr><tr><td>curved forceps</td><td>Roboz surgical instrument</td><td>RS-5137</td><td>Used for retina dissection</td></tr><tr><td>DDH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Elga LabWater</td><td>Elga PureLab Ultra</td><td>Used after ethanol to clean sonicator in between samples</td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>szX12</td><td>Used for retina dissection</td></tr><tr><td>Ethanol 200 proof</td><td>Decon laboratories</td><td>2701</td><td>To be used to clean sonicator in between samples</td></tr><tr><td>Foam floating tube rack</td><td>Thermo Fisher Scientific</td><td>36-099-2328</td><td>For tubes during incubation in water bath steps</td></tr><tr><td>General scissors</td><td>Roboz surgical instrument</td><td>RS-680</td><td>Used for retina dissection</td></tr><tr><td>Isoflurane 250 mL bottle</td><td>Piramal critical care</td><td>NDC &amp;nbsp;6679401725</td><td>Anesthesia</td></tr><tr><td>Isoflurane equipment</td><td>Vetequip sold by VWR</td><td>89012-492</td><td>Used to anesthetize prior to euthanasia</td></tr><tr><td>Kim wipes</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>Microplate reader</td><td>Molecular devices</td><td>SpectraMax M2 microplate reader</td><td>Used to read sample</td></tr><tr><td>Neurobasal- A media</td><td>Gibco</td><td>12349-015</td><td></td></tr><tr><td>Nose cone (low profile anesthesia mask)</td><td>Kent Scientific</td><td>SOMNO-0801</td><td>Used to deliver ansethesia</td></tr><tr><td>Objective on dissecting microscope</td><td>Olympus</td><td>DF plapo 1x pf</td><td>Used for retina dissection</td></tr><tr><td>Petri dish</td><td>VWR</td><td>25384-088</td><td>Used during retina dissection</td></tr><tr><td>Pierce assay reagent</td><td>Thermo Fisher Scientific</td><td>1861426</td><td></td></tr><tr><td>Pipette tips P20</td><td>Olympus Plastics</td><td>26-404</td><td></td></tr><tr><td>Pipette tips P200, P1000, P10 XL</td><td>VWR</td><td>76322-150, 76322-154, 76322-132</td><td></td></tr><tr><td>Pipetteman pipettes P200, P1000, P20, P10</td><td>VWR</td><td>F144055M, F144056M, F144058M, F144059M</td><td></td></tr><tr><td>SoftMax Pro software on computer</td><td>Molecular devices</td><td>SoftMax Pro 7 software</td><td>Software used to read sample</td></tr><tr><td>Sonic dismembrator</td><td>Thermo Fisher Scientific</td><td>FB50110</td><td>Sonicate sample (retina)</td></tr><tr><td>Transfer pipettes</td><td>Fisherbrand</td><td>13-711-9AM</td><td>Used to transfer retina from one tube to another</td></tr><tr><td>Vannas spring scissors</td><td>Katena</td><td>K4-5000</td><td>Used for retina dissection</td></tr><tr><td>Water bath set to 37 &amp;deg;C</td><td>N/A</td><td>N/A</td><td>Used for incubation</td></tr></tbody></table>

measuring uptake of the glucose analog, 6-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose, in intact murine neural retina

Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina

The retina is a highly metabolic tissue with multiple cell types requiring glucose and its derivatives to produce energy in the form of ATP. Retinal cells, including endothelial cells, neurons, photoreceptors, and glial cells, express glucose transporters (GLUTs; e.g., GLUT1-4) to enable the uptake of glucose for energy production. GLUT1 is the most abundantly expressed glucose transporter in the retina. This protocol enables researchers to measure the uptake of glucose in the neural murine retina in ex vivo conditions using the fluorescent glucose analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG). After retinal dissection, total retinal 6-NBDG levels can be easily determined via fluorescence endpoint measurement using a plate reader. For consistency, we recommend normalizing results to total protein levels. Although 6-NBDG is highly specific for GLUT1, uptake of this analog is detected in the presence of GLUT1 inhibitor BAY-876. As such, this assay provides a relatively quick and inexpensive method to measure glucose uptake ex vivo in whole mouse neural retina, which is partially mediated by GLUT1.

Figure 4 shows representative glucose fluorescence measurements from WT mouse retina incubated with 6-NBDG for different periods of time. After 30 min incubation, 6-NBDG levels were an average of 336 ± 27.91 AU, whereas after 60 min, levels of 6-NBDG increased to an average of 616.3 ± 8.38 AU. A further incubation of 30 min led to a reduced level of 6-NBDG (506.4 ± 5.3 AU). At 60 min, variability in the results was at a minimum and thus was chosen as the optimal time for incubation of the re...

Multiple cell types in the retina, including endothelial cells, neurons, and glial cells, express glucose transporters (GLUTs) to enable glucose uptake into cells. Using ex vivo mouse neural retina and the fluorescent glucose analog 6-NBDG, we describe a relatively quick and inexpensive method to measure glucose uptake in the whole mouse retina.

The retina is a highly metabolic tissue with multiple cell types requiring glucose and its derivatives to produce energy in the form of ATP. Retinal ...

measuring-uptake-glucose-analog-6-n-7-nitrobenz-2-oxa-13-diazol-4-yl

Research

JoVE Journal

Neuroscience

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

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.

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

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Learn to measure glucose uptake in intact murine neural retina using the fluorescent analog 6-NBDG. This ex vivo protocol enables rapid, cost-effective assessment of retinal metabolism for aging and disease research.

Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina (Video) | JoVE Journal

Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina

Explore More Videos