Name: cytosolic calcium measurements in renal epithelial cells by flow cytometry
Uploaded: 2014-10-28T13:00:00
Description: Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

Forskning i laboratorierne er finansieret af Fritz-Bender Foundation, München, Tyskland (TD) og en University of Witten / Herdecke interne forskningsbevilling (til W.-KL). Vi vil gerne takke Prof Dr. Dr. Frank Thévenod (Institut for Fysiologi, patofysiologi og Toksikologi, University of Witten / Herdecke) for nyttige forslag.

1. Fremstilling af reagenser og opløsninger <ol><li> Forberede en modificeret Hanks balancerede saltopløsning (HBSS) (i mM: 136,9 NaCI, 5,37 KCI, 0,34 Na 2 HPO 4, 0,44 KH 2 PO 4, 4,17 NaHCO3, pH 7,2, ingen phenolrødt). Opbevares ved 4 ° C. </li><li> Lav 1,5 M CaCl2 og 2 M 4- (2-hydroxyethyl) -1-piperazinethansulfonsyre (HEPES) (pH 7,2) stamopløsninger med destilleret vand. </li><li> Forbered en 250 mM stamopløsning af probenecid. For den frie syre...

<ol>
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Calcium er en vigtig begivenhed i flere cellulære processer, der virker som en anden messenger signalmolekyle og også som et middel til at kommunikere mellem ER og mitokondrier. Evnen til at måle ændringer i frie cytosoliske calciumkoncentrationer er en nyttig teknik, der kan anvendes på alle områder af cellebiologi. Der er forskellige metoder til at opdage cytosoliske calcium signaler. Klassisk, signaler fra en ratiometrisk calcium-indikator, såsom Fura-2, overvåges i levende celler ved hjælp af en fluorescens...

Calcium er en vigtig second messenger ansvarlig for transmission af cellulære fysiologiske og patofysiologiske signaler 1. Cytosoliske calciumkoncentrationerne holdes lavt gennem aktiviteten af ​​calcium pumper og calcium vekslere. Det sarkoplasmiske / endoplasmatiske reticulum calcium ATPase (SERCA) genopfylder det endoplasmatiske reticulum (ER) calcium lager som del af en &quot;pumpe lække&quot; system, hvorimod plasmamembranen calcium ATPase (PMCA) ekstruderer calcium til det ekstracellulære rum, både i ATP-afhængige måder 2. Fysiologiske messengers, såsom hormoner eller neurotransmittere, sender deres signaler gennem G-protein-koblede receptorer, aktivering af phospholipase C, der hydrolyserer phosphatidylinositol 4,5-bisphosphat (PIP2) i plasmamembranen at generere diacylglycerol og inositoltriphosphat (IP3) 3. Mens diacylglycerol forbliver i plasmamembranen, IP3 diffunderer ind i cytosolen og binder til IP3 receptors (IP3Rs), som ligand aktiverede calciumkanaler, fundet i ER-membranen og calcium frigives fra ER luminale butik kulminerede i en stigning i cytosoliske calciumkoncentrationer 4. En alternativ vej for calcium at nå cytosolen gennem calciumkanaler og varmevekslere til stede i plasmamembranen. Krydstale mellem disse to rum er blevet beskrevet: calcium induceret calcium frigivelse (CICR) hvor ekstracellulært calcium inducerer calcium frigivelse fra ER butikker 5, og butikken drives calcium post (SOCE) hvor tømning af ER stores registreres af STIM og forårsager åbning af Orai calciumkanaler i plasmamembranen at fremme genpåfyldning af ER-butikker 6. Under patofysiologiske tilstande, det cellulære calcium respons er deregulerede og cytosolisk calcium stigninger i fravær af fysiologiske stimulatorer 1. Calcium reaktion kan påvirkes på en række måder: langvarig calciumsignal, forsinket calcium fjernelse fra cytosolen, udtynding af calcium butikker eller lokaliserede ændringer i calcium. Endvidere mitokondrierne tage på en central rolle i buffering og frigive calcium 7. Langvarig og / eller supramaksimal cytosolisk calcium stigning fører til celledød. Faktisk calcium er oftere end ikke er involveret i den cellulære patofysiologiske respons og er en vigtig begivenhed i en bred vifte af sygdomme, såsom neurodegenerative sygdomme, hjertesygdomme, cancer, knoglesygdom og nyresygdom, som er hovedsagelig forbundet med celledød og tab eller ændring af organ eller væv funktion 8-10. Desuden er forstyrrelse af calcium signaler er knyttet til cellulær tilpasning og ændringer i cellulære funktioner og reaktioner. Traditionelt er calcium signaler målt med en negativt ladet fluorescerende calcium-indikator, der er lagt ind i celler i en celle-permeable acetoxymethyl (AM) ester form. Når spaltes af cellulære Esterases, forbliver fluorescerende indikatorer inden det intracellulære rum og stige i fluorescensintensitet, når calcium er bundet. Den mest kendte og anvendte calcium indikator er ratiometrisk Fura-2 er udviklet af Roger Tsien og kolleger 11. Calcium indikatorer med forskellige tilhørsforhold for calcium tillade forskellige calcium puljer, der skal overvåges. Detektionsmetoder inkluderer live-cell imaging, fluorescens mikropladeaflæser og flowcytometri. De relativt hurtige kinetik calcium signaler (normalt inden for et par sekunder til minutter) gør levende celle billeddannelse den bedste metode til at få de fleste oplysninger om karakteristika for calcium signal. Bortset fra de hurtige kinetik for calcium signaler (i millisekunder), live cell imaging er en bedre metode til at studere cellulære opdeling af calcium signalering inden for en enkelt celle 12. Absolutte calciumkoncentrationer kan beregnes ved at bestemme den minimale og maksimale fluorescens af calcium INDICATOR ved tilsætning af en calciumchelator og permeabilisering af cellerne, henholdsvis som beskrevet af Grynkiewicz et al. 11. Anvendelse af flowcytometri til måling af calcium signaler blev først udviklet i 1980&#39;erne i immunologiske celler, hvor muligheden for at måle flere parametre, såsom membran integritet og adskillelse af cellepopulationen, og kravet om celler i suspension kombineret med udviklingen af ​​enkelt bølgelængde calcium indikatorer gjort flowcytometri en ideel og convenientdetection metode 13-15. I adhærente celler, live cell imaging giver den mest information om calcium signalering, vigtigst kinetikken, men kræver en kompliceret opsætning, der omfatter et fluorescens mikroskop, et perfusionssystem, opretholdelse af det cellulære miljø, såsom temperatur, og specialiseret mikroskop software til at erhverve og analyse af data. Alternative metoder, såsom et fluorescens-mikropladelæser eller Pharmacological midler gennem brug af calciumchelatorer er uforlignelig i form af indsamlede informationer. Flowcytometri bliver mere udbredt at overvåge cytosolisk calcium i adhærerende celler dog i de fleste undersøgelser, kun endepunkt målingerne er udført. Brug den metode, der oprindeligt er udviklet af Gergely et al. I immunologiske B-celler 16 har ændringer i cytosol fri calciumkoncentration ved flowcytometri med real-time kinetik og overvågning af fluorescens intensitet i de enkelte celler fra en prøve population succes blevet målt i kræft epitelceller og cancer stamceller hybrider 17. Denne fremgangsmåde blev yderligere tilpasset til anvendelse i adhærerende epitelceller, som er vanskelige at indlæse med calcium indikatorer 18. Her, ved hjælp af en udødeliggjort klæbende epithelial cellelinie afledt fra S1 segmentet af rottenyre proximale tubulus (WKPT-0293 Cl.2) 19 beskriver vi en optimeret forenklet metode for målingen af ​​ændringer i cytosolisk calciumkoncentration ved flowcytometri. Da mange epitelceller i besiddelse af en række organiske transportører for anioniske forbindelser kan loading af celler med en calcium-indikator vise sig at være en udfordring. For at forhindre udstrømning af calcium indikatorer, probenecid, prototypisk inhibitor af organiske anion transportører og oprindeligt udviklet til at reducere renal udskillelse af penicillin 20, påføres samtidigt i inkubationsmediet. Cellerne opretholdes i monolag for calcium indikator lastning, bringes i suspension og calcium signaler kan påvises umiddelbart efter forbindelse tilsætning.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:79px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:321px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-3, AM</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">F-1241&#160;</td>
			<td>Cell permeable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Probenecid</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8761</td>
			<td>Water insoluble, dissolve in 1 N NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ionomycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thapsigargin</td>
			<td style="width:79px;">Tocris Bioscience</td>
			<td style="width:119px;">1138</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tunicamycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7765</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSCalibur Flow Cytometer + CELLQuest software</td>
			<td style="width:79px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Windows Multiple Document Interface for Flow Cytometry (WinMDI)</td>
			<td style="width:79px;">Joe Trotter, The Scripps Institute</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">WKPT-0293 Cl.2 rat kidney proximal tubule cell line</td>
			<td style="width:79px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Made available by Dr. Ulrich Hopfer (Department of Physiology &#38; Biophysics, Case 
			Western Reserve University, Cleveland, OH)</td>
		</tr>
	</tbody>
</table>

cytosolic calcium measurements in renal epithelial cells by flow cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

For at øge cytosolisk calcium, blev to farmakologiske forbindelser anvendes på renale proksimale tubuliceller (WKPT-0293 Cl.2) indlæst med celle gennemtrængelig calcium bindende farvestof, Fluo-3-AM. Ubehandlede kontrolprøver blev fyldt med calcium bindende farvestof i nærvær af probenecid og blev blandet, men uden tilsætning af nogen forbindelser. Thapsigargin (TG) er et klassisk inhibitor af SERCA pumpe fører til lækage ud af ER og resulterer i øget cytosolisk calcium. Tunicamycin (TUN) blokke N-glycosyleri...

Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Calcium er involveret i mange fysiologiske og patofysiologiske signalveje. Live-cell imaging kræver specialiseret udstyr og kan være tidskrævende. En hurtig, enkel metode under anvendelse af et flowcytometer til at bestemme de relative ændringer i cytosolisk calcium i adhærerende epitelceller bragt i suspension blev optimeret.

Cytosoliske Calcium Målinger i Nyrer epitelceller ved flowcytometri

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Research

JoVE Journal

Biology

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

Chromatin Isolering af RNA Oprensning (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Optimeret farvning og spredning modelleringsmetoder for celledeling Overvågning ved hjælp cellesporing Farvestoffer

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hurtig genetisk analyse af Epithelial-mesenkyme Signaling Under Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

High Efficiency Differentiering af humane pluripotente stamceller til hjertemuskelceller og karakterisering ved hjælp af flowcytometri

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Analyse af Cellesuspensioner isoleret fra Solid Væv af Spectral flowcytometri

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Måling af endoreduplikation ved flowcytometri af isolerede knold Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Høj overførselshastighed måling af Dictyostelium discoideum Macropinocytosis af Flow flowcytometri

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...