Name: a simple flow cytometric method to measure glucose uptake and glucose transporter expression for monocyte subpopulations in whole blood
Uploaded: 2016-08-12T14:00:00
Description: Watch this Scientific Journal Video about A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood at JoVE.com

Dit onderzoek werd gefinancierd door de Australische Centrum voor HIV en Hepatitis Virologie Research (ACH 2) en een 2010 ontwikkelingsstoornissen subsidie ​​(CNIHR) van de Universiteit van Washington Center for AIDS Research (CFAR), een NIH gefinancierd programma onder award nummer AI027757 die wordt ondersteund door de volgende NIH Institutes en Centers (NIAID, NCI, NIMH, Nida, NICHD, NHLBI, NIA). CSP is een ontvanger van de CNIHR en ACH 2 subsidie. SMC is een ontvanger van een National Health en Medical Research Council of Australia (NHMRC) Principal Research Fellowship. De auteurs zeer erkentelijk voor de bijdrage aan het werk van het Victoriaanse operationele infrastructuur Support Program door de Burnet Institute ontvangen. Wij erkennen de hulp van Geza Paukovic en Eva Orlowski-Oliver uit de AMREP Flow Cytometry Core Faciliteit voor flowcytometrie training en technisch advies. Wij danken Angus Morgan voor media-coaching en organisatie van de video shoot. onze dankJesse Masson en Jehad Abdulaziz K. Alzahrani voor lab begeleiding tijdens de video shoot. Wij danken de inspanningen van Dr. David Simar aan de School of Medical Sciences, UNSW, Australië die kritisch methodologisch advies aangeboden. CSP danken <a href="http://www.nice-consultants.com/">www.nice-consultants.com</a> voor grafische overleg. AUTEURS &#39;BIJDRAGE: CSP bedacht het project, ontworpen en experimenten, geanalyseerd en geïnterpreteerd data, en schreef het manuscript. JJA geïnterpreteerd gegevens en schreef het manuscript. TRB schreef het manuscript. JMM geïnterpreteerd data, maakte kritische intellectuele suggesties, en het manuscript beoordeeld. SMC interpreteren van gegevens, maakte kritische intellectuele suggesties en het manuscript beoordeeld.

LET OP: HIV-geïnfecteerde en HIV-geïnfecteerde patiënten werden gerekruteerd uit de Infectious Diseases Unit van The Alfred Hospital in Melbourne, VIC, Australië en uit de lokale gemeenschap, respectievelijk. Geïnformeerde toestemming werd verkregen van alle deelnemers, en het onderzoek werd goedgekeurd door het Comité Alfred Hospital Research and Ethics. 1. GLUT1 celoppervlak Detection op monocyten en monocyt subpopulaties <ol><li> Het verzamelen van bloed in citraat ACD-B antistoll...

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De hier beschreven protocol Gegevens een eenvoudige methode om glucose transporter expressie en fluorescerende glucose analoge opname door monocyten en monocyt subpopulaties in volbloed onderzocht. Door beoordeling 2-NBDG opname in volbloed, deze techniek maakt soortgelijke omstandigheden als in vivo. Een eerdere studie onderzocht 6-NBDG opname in monocyten gescheiden van volbloed door dichtheidscentrifugatie 17. Echter, deze studie niet onderzocht monocyten subpopulaties en scheiding van monocyten u...

Monocyten zijn een belangrijk onderdeel van de menselijke aangeboren immuunsysteem die snel gemobiliseerd naar plaatsen van infectie en ontsteking 1. Activatie van monocyten is kritisch voor het beperken van acute schade door ziekteverwekkers en is centraal in de pathogenese van een aantal chronische ziekten, waaronder atherosclerose 2, 3 kanker, HIV en 4,5. Het metabolisme van rustende en geactiveerde monocyten verschilt dramatisch, met rustende monocyten gebruik oxidatief metabolisme en geactiveerde monocyten gebruikmaking glycolytische stofwisseling (dwz fermentatie van glucose tot lactaat) 6. Activatie van monocyten induceert expressie van glucose transporters die zorgt voor verhoogde glucoseopname voor glycolytische stofwisseling 7. Monocyt glucose transporter 1 (GLUT1) is een dergelijke transporteur opgereguleerd tijdens activatie en zijn expressie is aangetoond dat leidt tot de productie van pro-inflammatoire cytokines in vitro en in vetweefsel van obese muizen 8. Infectie van een monocytische cellijn door Kaposi-sarcoom geassocieerd herpesvirus leidt tot cellulaire opregulatie van GLUT1 9, en we onlangs bleek dat tijdens chronische hiv-infectie een verhoogd percentage van GLUT1 uitdrukken monocyten tijdens onbehandeld en antiretrovirale therapie behandeld infectie 10 aanwezig zijn. Samengevat, deze studies tonen aan dat glucose en glycolytische metabolisme door monocyten zijn belangrijke aspecten van vele ontstekingsziekten. Dus een eenvoudige methode om monocyten GLUT1 expressie en glucose meten die homeostase en ontstekingsziekte waarschijnlijk van nut zijn voor een groot aantal onderzoekers. Menselijke monocyten zijn heterogeen, wordt samengesteld uit drie verschillende subsets die door differentiële expressie van het celoppervlak markers CD14 en CD16 11,12 kunnen worden onderzocht. Classical monocyten drukken een hoog niveau van CD14, maar niet uit te drukken CD16 (CD14 ++ CD16 -), tussenproduct monocyten een hoog niveau van CD14 en een tussenliggend niveau van CD16 (CD14 ++ CD16 +) en niet-klassieke monocyten een lage CD14 en een hoog niveau van CD16 (CD14 + CD16 ++). Monocyten die CD16 tot expressie zogenaamde CD16 + monocyten, die vergeleken met CD16 - monocyten hoge expressie van inflammatoire cytokines en het vermogen om beter te presenteren antigenen 13,14. Ongeveer 10% van de monocyten CD16 tijdens homeostase met hogere percentages waargenomen gedurende 15 inflammatie. Monocyt subpopulaties houden bepaalde ziektetoestanden en mogelijk bruikbare biologische markers van de ziekte en ziekteprogressie 16. Ons doel was om een ​​methode die glucose transporter expressie en glucose-opname door humane monocyten en monocyt subpopulaties onder omstandigheden zo dicht mogelijk bij phy kan meten identificerensiological voorwaarden mogelijk. Eerdere studies gemeten monocyten glucose transporter expressie en opname van glucose 17,18, hoewel deze methoden geïsoleerde monocyten die kunnen hebben veranderd eiwitexpressie in vergelijking met fysiologische omstandigheden 19 onderzocht, en geen eerdere studie heeft de menselijke monocyten subpopulaties onderzocht. Gebruik van multi-parametrische flowcytometrie beschrijven we een werkwijze glucose transporter expressie en opname van de fluorescente glucose analoge 2-NBDG van totale monocyten en monocyt subpopulaties onderzocht (op basis van CD14 en CD16 expressie) binnen het gehele ongemanipuleerd bloed.

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			<td height="61" style="height:61px;width:140px;">VACUETT Tube 9 ml ACD-B anticoagulant tubes</td>
			<td style="width:147px;">Greiner Bio-One GmbH</td>
			<td style="width:139px;">455094</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">5 ml sterile polypropylene tubes</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">352063</td>
			<td style="width:147px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:139px;">A7906</td>
			<td style="width:147px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:140px;">16% formaldehyde solution</td>
			<td style="width:147px;">Electron Microscopy Science</td>
			<td style="width:139px;">15710</td>
			<td style="width:147px;"></td>
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		<tr height="121">
			<td height="121" style="height:121px;width:140px;">BD FACS lysing solution (10X)</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">349202</td>
			<td style="width:147px;">Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4&#176;C)</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-CD3-PE</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">555340</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti CD14-APC</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">555399</td>
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		</tr>
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			<td height="22" style="height:22px;width:140px;">anti-CD16-PECy7</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">557744</td>
			<td style="width:147px;"></td>
		</tr>
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			<td height="22" style="height:22px;width:140px;">anti-Glut1-FITC</td>
			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">FAB1418F</td>
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			<td height="22" style="height:22px;width:140px;">IgG2b-FITC</td>
			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">IC0041F</td>
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			<td height="241" style="height:241px;width:140px;">2-NBDG</td>
			<td style="width:147px;">Life technologies</td>
			<td style="width:139px;">N13195</td>
			<td style="width:147px;">Suspend 5 mg of 2-NBDG into 1 ml of deionized water to make a 14.60 mM stock solution (keep for up to 6 months at 4&#176;C). To make the working 2-NBDG concentration, dilute stock 1/100 with 1X DPBS. Cover with foil. (store for up to 1 week at 4&#176;C)</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:140px;">Dulbecco&#8217;s Phosphate Buffered Saline (1X)</td>
			<td style="width:147px;">Life technologies</td>
			<td style="width:139px;">14190-144</td>
			<td style="width:147px;">To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4&#176;C)</td>
		</tr>
	</tbody>
</table>

a simple flow cytometric method to measure glucose uptake and glucose transporter expression for monocyte subpopulations in whole blood

Monocyten zijn aangeboren immuuncellen die kan worden geactiveerd door pathogenen en ontsteking geassocieerd met bepaalde chronische ontstekingsziekten. Activatie van monocyten induceert effectorfuncties en een gelijktijdige verschuiving van oxidatieve naar glycolytische stofwisseling die gepaard gaat met verhoogde glucose transporter expressie. Deze verhoogde glycolytische stofwisseling wordt ook waargenomen voor de geoefende immuniteit van monocyten, een vorm van aangeboren immunologisch geheugen. Hoewel in vitro onderzoek protocollen glucose transporter expressie en glucose opname door monocyten zijn beschreven, zijn geen onderzocht door multi-parametrische flowcytometrie in volbloed. We beschrijven een multi-parametrische flowcytometrische protocol voor het meten van fluorescentie glucose analoge 2-NBDG opname in volbloed door totale monocyten en de klassieke (CD14 ++ CD16 -), tussenproduct (CD14 ++ CD16 +) en niet-klassieke ( CD14 + CD16 ++) monocytsubpopulaties. Deze methode kan worden gebruikt om glucose transporter expressie en glucoseopname voor totale monocyten en monocyt subpopulaties bij homeostase en ontstekingsziekten onderzocht en kunnen eenvoudig worden aangepast om glucoseopname andere leukocyten en leukocyten subpopulaties binnen bloed onderzocht.

Compensatie moet worden uitgevoerd voor individuele fluorochromen fluorescentie spillover voorkomen. Monocyten worden eerst verrijkt door venstertijd gebaseerd op voorwaartse en zijwaartse verstrooiing. De plots gepresenteerde vertegenwoordigers van ten minste zes onafhankelijke experimenten uitgevoerd op volbloed dat bij zes of meer deelnemers zoals eerder beschreven 10 Figuur 1A toont de eerste gating van monocyten door cel verstrooiing en uitsluiting van T-...

Watch this Scientific Journal Video about A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood at JoVE.com

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Een eenvoudige Flowcytometrische methode om te meten glucose opname en Glucose Transporter Expression voor monocyten subpopulaties in volbloed

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

The overall goal of this methodology is to measure the uptake of glucose and its transporter on different monocyte subpopulations. This method can be used to answer key questions in the immunology field, such as What is the importance of immune cell metabolism in disease outcomes? The main advantages of these techniques are it is quick, easy to perform, requires no reactive material, and requires only a small volume of blood.Within one hour of collecting the blood samples, in citrate ACD-B anticoagulant tubes, place the samples into a biological safety cabinet. Next, add 100 microliters of blood from each sample into individual polypropylene tubes, and treat the cells with two milliliters of 1X lysing solution on ice, pipetting gently to mix. After 15 minutes, centrifuge the cells and follow this with two more spins in wash buffer.After the second wash, use a pipette to carefully remove as much of the supernatants as possible, and re-suspend the pellets in 100 microliters of wash buffer on ice. To identify the specific monocyte subpopulations, stain the cells with five microliters of the appropriate antibodies per 100 microliters of cells, for 30 minutes on ice in the the dark. At the end of the incubation, wash the samples two times in wash solution and fix the cells with 200 microliters of 0.5%formaldehyde in PBS.Then, analyze the cells within 24 hours on a flow cytometer capable of detecting four colors. To assess the glucose uptake of the monocyte subpopulations, transfer 90 microliters of blood from each sample into individual polypropylene tubes, and add 10 microliters of the fluorescent glucose analog of interest to the blood. Flick the tubes gently to mix, and cover the samples with aluminum foil.Then, place the cells at 37 degrees Celsius for 15 to 30 minutes, followed by their immediate transfer to ice. Next, add four milliliters of FACS lysing solution to the tubes, and centrifuge the cells. Then, perform a second centrifugation in wash buffer.Decant the supernatant, and stain the cells with the appropriate antibodies of interest for 30 minutes at four degrees Celsius in the dark. Then, wash the samples in four milliliters of ice-cold wash buffer, re-suspend the pellets in 200 to 300 microliters of ice-cold PBS, and analyze the tubes within 10 minutes. For flow cytometric analysis, first use the unstained and individually stained control samples to set the compensation.Next, in the appropriate single-cell analysis software, open the first sample in a forward by side scatter plot, and gate the monocytes as illustrated. Then, double-click the gated monocyte population, and gate the CD3-negative population. Now double-click on the CD3-negative population, and set the x-axis to display the CD14 staining, and the y-axis to display the CD16 staining.Next, to measure the uptake of glucose or its transporter in a specific monocyte subpopulation, generate the appropriate histograms. Then, where defined cell populations exist, use the control FITC igG samples to set the gate and determine the total percentage of CD14-positive monocytes in each sample. Here, the initial gating strategy for isolating the monocytes by cell scatter and T-cell exclusion by gating within the CD3-negative population, is shown.The monocytes are then gated for CD14 expression alone, or in combination with CD16 to identify the total monocytes or monocyte subpopulations. Distinct populations of CD14-positive Glut-1 glucose transporter positive monocytes may be observed, most notably in the circulating white blood cells obtained from HIV-positive individuals. CD14-positive Glut-1 positive cells may also be observed within specific monocyte subsets in HIV-uninfected individuals, although these populations are more pronounced in HIV-positive patients.As expected from the glucose transporter expression data, the glucose analog 2-NBDG, is also taken up in higher quantities by cells from HIV-positive individuals. Once mastered, this technique can be completed within one-and-a-half hours if performed properly. While attempting this procedure, it is important to limit light exposure to all regions.Following glucose analog uptakes, cells can be stained with cell surface antibodies to identify specific immune populations that you're interested in. After its development, this method can be used to determine how HIV affects immune cell metabolism, can also be explored by other researchers, in different fields, such as immunology, virology, and cancer research. After watching this video, you should have a good understanding on how to detect glucose transporter one, and glucose uptake in immune cells.Don't forget that when working with infectious agents, standard safety procedures should be employed.

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