Name: staining the cytoplasmic ca2+ with fluo-4/am in apple pulp
Uploaded: 2021-11-06T13:00:00
Description: Watch this Scientific Journal Video about Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp at JoVE.com

Dette arbeidet ble støttet av Agricultural Variety Improvement Project of Shandong Province (2019LZGC007) og Fruit tree innovasjonsteam av Shandong moderne landbruksindustriteknologisystem (SDAIT-06-05).

1. Protoplast ekstraksjon
<ol><li>Forbered den grunnleggende løsningen: 20 mM CaCl2,5 mM 2-(N-morpholino)etanesulfonsyre og 0,4 M D-sorbitol. MERK: pH i grunnløsningen ble justert til 5,8 med 0,1 M Tris buffer, filtrert gjennom 0,22 μm vannløselige filtre og lagret ved 4 °C.</li>
	<li>Forbered den enzymatiske løsningen: Bland 0,3% (w / v) Macerozyme R-10 og 0,5% (w / v) cellulase R-10 med den grunnleggende løsningen.</li>
	<li>Tilsett 0,5 ml enzymatisk oppløsning i et 1,5 ml sentr...

<ol>
	<li>Hocking, B., Tyerman, S. D., Burton, R. A., Gilliham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+calcium:+Transport+and+physiology.">Fruit calcium: Transport and physiology.</a> Frontiers in Plant Science. 7, 569 (2016).</li><li>Li, J., Yang, H. -. q., Yan, T. -. l., Shu, H. -. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+indole+butyric+acid+on+the+transportation+of+stored+calcium+in+Malus+hupehensis+rhed.+Seedling.">Effect of indole butyric acid on the transportation of stored calcium in Malus hupehensis rhed. Seedling.</a> Agricultural Sciences in China. 5 (11), 834-838 (2006).</li><li>Gao, Q., Xiong, T., Li, X., Chen, W., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+calcium+sensors+in+fruit+development+and+ripening.">Calcium and calcium sensors in fruit development and ripening.</a> Scientia Horticulturae. 253, 412-421 (2019).</li><li>Barrett, D. M., Beaulieu, J. C., Shewfelt, R. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+availability+regulates+the+development+of+root+hairs+in+Arabidopsis+thaliana+(L.)+Heynh.">Magnesium availability regulates the development of root hairs in Arabidopsis thaliana (L.) Heynh.</a> Plant Cell and Environment. 37 (12), 2795-2813 (2014).</li><li>Qiu, L., Wang, Y., Qu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading+calcium+fluorescent+probes+into+protoplasts+to+detect+calcium+in+the+flesh+tissue+cells+of+Malus+domestica.">Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica.</a> Horticulture Research. 7, 91 (2020).</li><li>Boyer, J., Liu, R. 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I denne studien ble levedyktige protoplaster oppnådd ved enzymatisk hydrolyse. Vær oppmerksom på at denne metoden krever friske epler. Den nåværende protokollen muliggjør rask isolering av et stort antall protoplaster fra fruktmasse til bruk i forskningsstudier. Anvendelsen av denne metoden er ikke begrenset til 'Fuji'; protoplastene til eplemassen til 'Dounan' og 'Honey Crisp' kan også ekstraheres gjennom samme protokoll (Supplerende figur S4). Protoplastløsningen etter enzymolyse inneholder cel...

Ca2+ spiller en viktig rolle i plantesignaltransduksjon og metabolisme1,2. Videre regulerer det fruktkvalitetsegenskaper3,4, inkludert hardhet, sukkerinnhold og følsomhet for fysiologiske lidelser under lagring5,6. Cytoplasmic Ca2 + spiller en viktig rolle i signaltransduksjon og regulerer plantevekst og utvikling7. Forstyrrelse av cellulær kalsium homeostase kan indusere bitter grop i epler8, brun flekk sykdom i pærer9, og navlestreng rot i tomater10, påvirker fruktkvaliteten og forårsaker alvorlige økonomiske tap3,11. Kalsiumavbildning har tilstrekkelig romlig og tidsmessig oppløsning og er en viktig metode for å observere Ca2 + dynamikk i levende celler12,13.
For tiden er det to hovedmetoder for intracellulær kalsiumavbildning i levende celler: den ene bruker kjemiske småmolekylære fluorescerende sonder14, og den andre er genkodingssensoren (GECI)15,16. Gitt vanskeligheten med å etablere et stabilt transgent system i frukttrær og lengre fruktutvikling, er GECIS uegnet for frukt Ca2 + fluorescensavbildning.
Småmolekyl fluorescerende sonder som Fluo-4 / AM har en spesiell fordel: deres AM-esterform (cellegjennomtrengelig acetoksymetylsterderivat) kan lett bulkbelastes i levende celler uten behov for transfeksjon, noe som gjør det fleksibelt, raskt og ikke-cytotoksisk17. Fluo-4 / AM kunne med hell lastes inn i pollenrøret i Pyrus pyrifolia18 og Petunia,19 samt inn i vaktceller20 og rothår av Arabidopsis21.
For tiden er det få rapporter om kalsiumfluorescensfarging av masseceller22. Som et viktig mineralelement spiller kalsium en nøkkelrolle i veksten og kvalitetskontroll av trefrukter som epler. Epletrær er globalt anerkjent som en viktig økonomisk art, og epler regnes som en sunn mat23. I denne studien fikk vi levedyktige protoplaster fra eplefruktmasse gjennom enzymatisk hydrolyse og lastet deretter småmolekylet fluorescerende reagenser inn i cytoplasma for å oppdage Ca2+.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10&#215; phosphate-buffered saline</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td>PBS (phosphate buffered solution) is a phosphate buffer solution, which can provide a relatively stable ionic environment and pH buffering capacity. It is a buffer salt solution often used in biology for molecular cloning and cell culture. The pH is 7.4.&#160;</td>
		</tr>
		<tr>
			<td>2-(N-morpholino)ethanesulfonic acid</td>
			<td>Solarbio</td>
			<td>M8010</td>
			<td>Biological buffer</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Solarbio</td>
			<td>C8370</td>
			<td>Calcium chloride dihydrate is a white or gray chemical, mostly in granular form.</td>
		</tr>
		<tr>
			<td>Cellulase R-10</td>
			<td>Yakult Honsha</td>
			<td>MX7352</td>
			<td>Degrade plant cell walls.</td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Solarbio</td>
			<td>S8090</td>
			<td>It has good moisturizing properties, prevents drying, and prevents sugar, salt, etc. from crystallizing.</td>
		</tr>
		<tr>
			<td>F-127</td>
			<td>Thermo Fisher Scientific</td>
			<td>P6867</td>
			<td>Pluronic F-127 is a non-ionic, surfactant polyol (molecular weight of approximately 12500 Daltons), which has been found to be beneficial to promote the dissolution of water-insoluble dyes and other materials in physiological media.&#160;</td>
		</tr>
		<tr>
			<td>FDA</td>
			<td>Thermo Fisher Scientific</td>
			<td>F1303</td>
			<td>FDA is a cell-permeant esterase substrate that can serve as a viability probe that measures both enzymatic activity, which is require to activate its fluorescence, and cell-membrane integrity, which is required for intracellular retention of their fluorescent product.&#160;</td>
		</tr>
		<tr>
			<td>Fluo-4/AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>The green fluorescent calcium indicator Fluo-4/AM is an improved version of the calcium indicator Fluo-3/AM. The Fluo-4/AM loads faster and is brighter at the same concentration. It can be well excited with a 488 nm argon ion laser.</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Thermo Fisher</td>
			<td>EVOS Auto 2</td>
			<td>Observe the fluorescence image.</td>
		</tr>
		<tr>
			<td>Macerozyme R-10</td>
			<td>Yakult Honsha</td>
			<td>MX7351</td>
			<td>Degrade plant tissue to separate single cells.</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td>It is widely used in the preparation of buffers in biochemistry and molecular biology experiments.</td>
		</tr>
	</tbody>
</table>

staining the cytoplasmic ca2+ with fluo-4/am in apple pulp

Cytosolic Ca2+ spiller en nøkkelrolle i planteutvikling. Kalsiumavbildning er den mest allsidige metoden for å oppdage dynamiske endringer i Ca2+ i cytoplasma. I denne studien fikk vi levedyktige protoplaster av masseceller ved enzymatisk hydrolyse. Isolerte protoplaster ble inkubert med det lille molekylet fluorescerende reagens (Fluo-4/AM) i 30 min ved 37 °C. De fluorescerende sondene farget vellykket cytosolic Ca2 +, men akkumulerte ikke i vakuoler. La3+, en Ca2+ kanalblokker, redusert cytoplasmatisk fluorescensintensitet. Disse resultatene tyder på at Fluo-4/AM kan brukes til å oppdage endringer i cytosolisk Ca2+ i fruktkjøttet. Oppsummert presenterer vi en metode for effektivt å isolere protoplaster fra kjøttceller av frukten og oppdage Ca2 + ved å laste inn et lite molekyl kalsiumfluorescerende reagens i cytoplasma av masseceller.

Etter protokollen beskrevet ovenfor brukte vi den enzymatiske metoden for å oppnå levedyktige protoplaster fra massen (figur 1). Noen protoplaster hadde vakuoler, mens andre ikke gjorde det. Mens protoplastene ikke viste fluorescens da Ca2 + fluorescerende indikator ikke ble lastet inn i dem. Da Fluo-4/AM ble lastet inn i protoplastene, ble cytoplasma, men ikke vakuolen, fluorescerende (figur 2). Dette resultatet indikerte at Fluo-4 / AM vellykket f...

Watch this Scientific Journal Video about Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp at JoVE.com

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp

Isolerte protoplaster av eplemasseceller ble lastet med et kalsiumfluorescerende reagens for å oppdage cytoplasmatisk Ca2 + konsentrasjon.

Farging av Cytoplasmic Ca2+ med Fluo-4/AM i Apple Pulp

Cytosolic Ca2+ plays a key role in plant development. Calcium imaging is the most versatile method to detect dynamic changes in Ca2+ in the cytoplasm. In ...

Som et viktig mineralelement spiller kalsium en nøkkelrolle i veksten og kvalitetskontroll av trefrukter som eple, det kan regulere hardheten, sukkerinnholdet, lagringsperioden og den fysiologiske lidelsen under lagring. Kalsiumavbildning er den mest vestlige metoden for å oppdage dynamiske endringer i kalsiumioner i cytoplasma. Små molekylfluorescerende indikatorer, som fluo-4/AM har kalsiumionspesifikk selektivitet og kan ikke-invasivt lastes av esterifiseringsinkubering, som er fleksibel, rask og ikke-cytotoksisk.Denne protokollen vil introdusere en metode for lasting av små molekyl kjemiske fluorescerende reagensfluo-4/AM i planteprotoplaster. Protoplast ekstraksjon Tilsett 0,5 milliliter enzymoppløsning i et sentrifugerør på 1,5 milliliter. Velg et sunt og modent eple, og skjær deretter massen i 10 ganger 5 ganger 1 kubikk milliliter størrelse.Legg massestykkene i enzymatisk løsning, og lukk deretter røret. Inkuber røret ved 28 grader i en time. Lenket ved 70 o/min i mørket.Deretter vasker du massestykkene, aspirerer all den enzymatiske løsningen og tilsett deretter 0,5 milliliter grunnleggende løsning. Sentrifuge med en hastighet på 300 g i 2 minutter. Protoplasts suspensjon kan oppnås ved å aspirere bunnløsningen.Småmolekyl kjemisk fluorescens reagensfarging. Forbered fluo-4/AM-lasteløsningen med en 2 millimolar fluo-4/AM, 20% etyl127 og den 10X fosfatbufrede saltoppløsningen i forholdet en til en til en til to. Tilsett 1 mikroliterlasteløsning av fluo-4/AM til 99 mikroliter protoplast suspensjon.Den endelige konsentrasjonen av fluorescerende fargestoffer er 5 mikromolar. Bland løsningen og lukk deretter røret. Samtidig stryker lanthanum kalsium eller unikhet av det lille molekylære kjemiske fluorescerende middelet.Tilsett en mikroliter eller 10 mikroliter mer lanthanum jern og deretter en mikroliter lasteløsning av fluo-4/AM til 98 mikroliter protoplaster suspensjon. Den endelige konsentrasjonen av lanthanum jern er 100 mikromolar. Bland løsningen og lukk deretter røret.Inkuber ved 37 grader i 30 minutter i mørket. Deretter sentrifugerer du med en hastighet på 300 G i to minutter. Aspirer til 17 mikroliter oppløsning og tilsett en 17 mikroliter grunnleggende løsning.Inkuber protoplastfjæringen ved 37 grader i 30 minutter for å helt Etter det aspirerer du 50 mikroliter protoplaster suspensjon og drypper videre til et lysbilde. Vær oppmerksom på mikroskopet for fluorescens. Velg 20 ganger forstørrelsen og GFP-kanalen du vil observere.Juster lysstyrken jevnt til 0,5. Et kritisk skritt i denne foreslåtte metoden er å vaske flekken i protoplastene og inkubere dem i 30 minutter. Utilstrekkelig vask forårsaker overdreven rygg på fluorescensen under observasjon og inkubasjon gir bedre lasting eller sonden inn i cytoplasma.Protoplaster Levedyktighet Analyse Ta 99 mikroliter protoplaster suspensjon etter inkubering ved 37 grader i 30 minutter. Tilsett 1 ml ved FD-oppløsningen til protoplasts suspensjon. Bland oppløsningen ved å pipettere opp og ned flere ganger og lukk deretter røret.Hold deg ved romtemperatur i fem minutter i mørket. Forbered lysbildene og observere dem på fluorescensmikroskopet med GFP-kanal. Vi presenterer resultatene for deg etter figur én.Vi bruker den enzymatiske metoden som alle får protoplaster fra massen. Noen protoplaster hadde en vakuol, mens andre ikke gjorde det. Protoplastene ble farget med FD i fem minutter og viser florescerende i cytoplasma som indikerer at høy temperatur, 37 grader ikke påvirker levedyktigheten eller protoplastene figur to.Protoplastene viste ingen fluorescens i en av kalsiumjernenes fluorescensindikator, eller den er ikke lastet inn i dem. Enn i fluo-4/AM ble lastet inn i protoplasten, ble cytoplasten, men ikke en vakuol fluorescerende. Lanthanum Irons som effektivt blokkerer kalsiumkanaler er cellemembranen ble lagt til Protoplast fluo-4 / AM lasting.Ved den endelige konsentrasjonen av 100 mikromolar lanthanum jern reduserer kalsiumfluorescensintensiteten betydelig. Dette resultatet viste videre at fluo-4/AM effektivt holder seg i kalsiumjernene i cytoplasten, og det viser dynamiske endringer i kalsiumjerninnholdet. Figur tre Fluo-4/AM-resultatene ble analysert ved hjelp av IMH-godkjent pluss 6.0 programvare fluo-4/AM farging med tilsetning av lanthanum jern reduserte florescensverdien av cytoplasmatiske kalsiumjern betydelig.I denne videoen ble alle protoplaster oppnådd ved enzymatisk hydrolyse. Vi lastet fluorescerende sonder inn i en protoplast ved 37 grader. Mer over, metoden påvirket ikke levedyktigheten til protoplaster.Oppsummert er metoden beskrevet her i detalj. De dynamiske endringene i cytoplasmatiske kalsiumioner i eplemasseceller gir dermed 10 like støtter for videre formål med kalsiumrelaterte studier.

Research

JoVE Journal

Biology

Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp

Focal Ca2+ Transient Detection in Smooth Muscle

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Automatisert analyse av Dynamic Ca<sup&gt; 2 +</sup&gt; Signaler i bildesekvenser

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulering av cytoplasmatiske DNA Sensing Pathways<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Lokal Ca<sup&gt; 2+</sup&gt; Signaler i dyrkede pattedyrceller

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Metoder for klassifisering av cytoplasmisk foci som mammalstresskorn

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...