Name: mammalian cell division in 3d matrices via quantitative confocal reflection microscopy
Uploaded: 2017-11-29T14:00:00
Description: Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Dette arbejde blev støttet af NIH grants R01CA174388 og U54CA143868. Forfatterne vil gerne anerkende PURA award fra Johns Hopkins University for støtte af Wei-tong Chen. Dette materiale er baseret på arbejde støttes af de National Science Foundation Graduate Research Fellowship under Grant No. 1232825.

Protokollen i henhold følger retningslinjerne for The Homewood institutionelle Review Board (HIRB).
1. stabil udtryk for H2B-mCherry som markør for celle mitose
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Generation af lentiviral partikler fra menneskelige embryonale nyre 293T (HEK 293T) celler
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<li>Plade HEK 293T celler på en 10 cm celle kultur parabol med tæthed på 5 x 106 celler/parabol i cellekulturmedium (Dulbeccos modificerede Eagle's Medium (DMEM) indeholder høje glukose (4,5 g/L)...

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Den forrige undersøgelse af celledeling i 3D var ikke effektivt på grund af eksperimentelle begrænsninger og tekniske udfordringer18,19. De kritiske trin for effektiv undersøgelse af pattedyr celledeling i 3D kollagen matricer er: (1) indarbejdelsen af fluorescens-mærket mitotiske markører til celler; (2) synkroniseringen af celledeling; og (3) overvågning af division begivenheder i 3D matricer ved hjælp af live-celle imaging teknik, tidsopløst fluoresce...

Celle mitosen er en afgørende begivenhed i cellulære liv, regulering af som spiller afgørende roller i væv og orgel udvikling. Unormal mitosen er impliceret i naturlige genetiske variationer, menneskelige aldringsprocesser og progression af kræft1,2,3,4,5. Den forhøjede sats af udbredelsen af tumorceller sammenlignet med normale celler er et af kendetegnene ved kræft, trods at celle adfærd er ganske heterogene blandt forskellige typer af tumorer, og selv blandt patienter. På trods af lovende prækliniske resultater, har nogle nyligt udviklede antimitotic lægemidler ikke vist sig for at være effektiv i kliniske forsøg6,7,8,9,10 ,11. Relevansen af eksperimentelle og prækliniske modeller må betragtes. Mange typer af normale pattedyr og kræft celler opdele i tre-dimensionelle (3D) matricer, såsom fibroblaster og fibrosarcoma celler i kollagen-rige 3D bindevæv og metastatisk kræftceller i den 3D stromale ekstracellulære matrix (ECM). Dog, har størstedelen af pattedyr celledeling eksperimenter og assays udført på celler dyrkes på todimensionelle (2D) substrater. En manipuleret 3D matrix kunne bedre sammenfatte mikrostruktur, mekaniske egenskaber og biokemiske signaler af 3D ECM af både normale og patologiske væv12,13,14, 15,16,17.
Undersøgelse af hvordan pattedyr celledeling er reguleret i 3D miljøer forbliver stort set uudforskede trods både den fysiologiske relevans og terapeutiske betydning18,19. Mulige årsager omfatter de tekniske vanskeligheder og eksperimenterende udfordringer forbundet med at studere celledeling i 3D matricer. Celle mitosen udgør en lille tidsmæssige brøkdel i hele cellecyklus20. Tidligere arbejde har vist, at spredning satsen af mange pattedyrceller, såsom menneskelige bryst adenocarcinom MCF-7, menneskelige osteosarkom U2OS og menneskelige leveren HepG2, er meget lavere i 3D matricer sammenlignet med deres modstykker på 2D substrater21, 22. Derudover flytte celler indlejret i 3D matricer ind og ud af fokus under live-celle billeddannelse. Alle disse faktorer bidrager til den ekstremt lav effektivitet indfange celledeling begivenheder i 3D kultur ved hjælp af Billeddannende teknikker.
Interaktioner mellem ECM og celler spiller en kritisk rolle i regulering celledelinger. Her, beskriver vi en fremgangsmåde til at effektivt studere pattedyr celledeling i 3D kollagen matricer. Metoden omfatter indarbejdelse af mitotiske markører til cellerne, synkronisering af celledeling, samt overvågning af division begivenheder i 3D matricer ved hjælp af live-celle imaging teknik, tidsopløst fluorescens refleksion mikroskopi, og kvantitative imaging analyse. Fluorescens-mærket histoneprotein H2B føres først ind i cellerne som en markør for at skelne mitotiske og interphase celler. Derefter cellerne synkroniseres ved hjælp af en kombination af thymidine blokering og nocodazole behandling, efterfulgt af en mekanisk shake-off teknik. Synkroniseret celler er derefter direkte indkapslet i 3D kollagen matricer. Celledeling begivenheder af flere celler overvåges effektivt ved hjælp af lav-forstørrelse time-lapse live-celle billeddannelse. Deformationen af kollagen fibre, som er en indikator for celle-matrix interaktion, overvåges ved hjælp af mikroskopi Konfokal refleksion på høj forstørrelse.
Vi har tidligere brugt denne teknik til at overvåge og kvantificere celle-matrix interaktion før, under og efter mitosen to metastatisk kræft cellelinjer, menneskelige invasive duktalt carcinom MDA-MB-231 og menneskelige fibrosarcoma HT1080 celler, i 3D kollagen matricer19. De metoder, der præsenteres her giver en effektiv og generel tilgang for at studere begge pattedyr celledeling i et 3D miljø og celle-matrix interaktioner. MDA-MB-231 cellelinie bruges som forbillede i hele papiret. Denne protokol giver ny indsigt i de molekylære grundlag for udviklingen af normale væv og sygdomme, og kan også give mulighed for udformningen af nye diagnostiske og terapeutiske strategier.

<table>
	<tbody>
		<tr>
			<td>Human embryonic kidney 293T</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>Physical Sciences Oncology Center, NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>ThermoFisher Scientific</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life technologies</td>
			<td>11668-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding H2B-mCherry in a lentiviral vector</td>
			<td>Addgene</td>
			<td>plasmid 21217</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>GibcoBRL</td>
			<td>11810-025</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>113375-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Bake</td>
			<td>3722-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV syringe filter unit, 0.45-&#956;m, PVDF, 33 mm</td>
			<td>Millipore</td>
			<td>SLHVM33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000E epifluorescence microscope</td>
			<td>Nikon</td>
			<td>TE2000E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 1K CCD camera</td>
			<td>Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1 confocal microscope</td>
			<td>Nikon</td>
			<td>A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammalian cell division in 3d matrices via quantitative confocal reflection microscopy

Undersøgelse af hvordan pattedyr celledelingen reguleres i et 3D miljø forbliver stort set uudforskede trods dens fysiologiske relevans og terapeutiske betydning. Mulige årsager til manglen efterforskning er eksperimenterende begrænsninger og tekniske udfordringer at gør studiet af celledeling i 3D kultur ineffektive. Her, beskriver vi en imaging-baseret metode til effektivt studere pattedyr celledeling og celle-matrix interaktioner i 3D kollagen matricer. Celler mærket med fluorescerende H2B synkroniseres ved hjælp af en kombination af thymidine blokering og nocodazole behandling, efterfulgt af en mekanisk shake-off teknik. Synkroniseret celler er derefter indkapslet i en 3D kollagen matrix. Celledeling overvåges ved hjælp af live-celle mikroskopi. Deformation af kollagen fibre under og efter celledeling, som er en indikator for celle-matrix interaktion, kan overvåges og kvantificeres ved hjælp af kvantitative Konfokal refleksion mikroskopi. Metoden giver en effektiv og generel tilgang for at studere pattedyr celledeling og celle-matrix interaktioner i en fysiologisk relevante 3D miljø. Denne tilgang ikke blot giver ny indsigt i de molekylære grundlag for udviklingen af normale væv og sygdomme, men også mulighed for udformningen af nye diagnostiske og terapeutiske strategier.

Målet med denne artikel er at fremlægge en imaging-baseret metode til at studere pattedyr celledeling processer i 3D matricer og kvantificere interaktioner mellem cellen og 3D ekstracellulære matrix, under og efter celledeling. For at lette billedbehandling i celle mitosen, indarbejdet vi H2B-mCherry i MDA-MB-231 celler ved hjælp af lentiviral transduktion. H2B konjugeret med fluorescerende proteiner er brugt som mitotiske markør til at skelne mitotiske celler fra interphase celler o...

Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

Denne protokol effektivt undersøgelser pattedyr celledeling i 3D kollagen matricer ved at integrere synkronisering af celledeling, overvågning af division begivenheder i 3D matricer ved hjælp af live-celle imaging teknik, tidsopløst fluorescens refleksion mikroskopi og kvantitative imaging analyse.

Pattedyr celledeling i 3D matricer via kvantitative Konfokal refleksion mikroskopi

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

The overall goal of this procedure is to study mammalian cell division and cell matrix interactions in a physiologically relevant 3D environment. The main advantage of this technique is that it provides an efficient and general approach to study mammalian cell division and cell matrix interactions. This method can help answer key questions in cell biology such as the molecular basis of the development of normal and disease tissue.A day prior to transfection, plate five million HEK-293 T-cells in a 100 milliliter cell culture dish in 10 milliliters of normal cell culture medium. Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day just before starting transfections, remove a vial of the transfection reagent from four degrees Celsius and leave it out to reach room temperature.Then to start, add one milliliter of reduced serum medium to a sterile microfuge tube. To this, add specified amounts of lentiviral vector plasmids used for transfection and mix by pipetting. Incubate the tube at room temperature for five minutes.After five minutes, add 48 microliters of the transfection reagent into this tube and mix gently by pipetting. Subsequently, incubate this mixture at room temperature for at least 15 minutes. Once the incubation is complete, remove the plate with HEK-293 T-cells from the incubator.Add the prepared DNA transfection reagent mixture drop wise onto the cells and gently swirl the plate to ensure even distribution in the plate. Place the plate back into the incubator. Approximately six hours after transfection, remove the plate and aspirate the medium.Add 10 milliliters of fresh culture medium and transfer the plate back into the incubator. At multiple time points, 24, 48, and 72 hours post-transfection, harvest the culture medium containing the lentiviral particles in separate tubes. Next, filter the harvested culture medium through a sterile 0.45 micron filter to remove cellular debris.Replace with fresh culture medium following each harvest. The filtrate containing the lentiviral particles can be used for transduction right away or can be stored at minus 80 degrees Celsius. Plate human breast carcinoma cells MDA-MB-231 in a 35 millimeter culture dish in normal culture medium.Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day, aspirate the culture medium from the plate and add one milliliter of the lentiviral particles together with one milliliter of fresh culture medium. Incubate the cells in the incubator for about 16 hours.After the incubation with viral particles is complete, remove the plate to aspirate the medium. Then add two milliliters of fresh medium and transfer back to the incubator for 24 to 72 hours. Check the expression of H2B-mCherry in these transduced cells using an epifluorescence microscope.To start the synchronization experiments, first plate MDA-MB-231 cells expressing H2B-mCherry at 50 to 60%confluency in a 24-well plate. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, replace the culture medium with 0.5 milliliters of medium containing two millimolar Thymidine.Leave the plate in the incubator for 24 hours. Once the incubation is complete, wash the cells three times with PBS to release them from the Thymidine treatment. Then leave the cells in 0.5 milliliters of normal culture medium for five hours in the incubator.After this, to block these cells in mitosis, add Nocodazole to the culture medium. Leave the cells in the incubator for 12 hours. After the Nocodazole treatment is complete, take out the plate and using an orbital shaker, shake the cells for 45 seconds to one minute to release mitotic cells.Subsequently, transfer the medium containing the extracted mitotic cells into a new centrifuge tube. Then add 0.5 milliliters of fresh medium to each well. After, repeat the shaking and extraction of cells two more times.Pool the total collected medium and then centrifuge it to pellet the mitotic cells. After the spin, resuspend the cell pellet in 0.25 milliliters of fresh cell culture medium. Using a hemocytometer, count the number of cells.At the start of this step, prepare 50 milliliters of 10x DMEM solution and filter sterilize it. Next, determine the volumes of all the components that will be used to make the 3D collagen matrix as specified in the text protocol. Pre-chill all these components on ice to slow down the gelation of collagen.Now, to a pre-chilled 1.5 milliliter centrifuge tube, add the required numbers of cells in medium then 10x DMEM, FBS, deionized water, collagen, and finally sodium hydroxide. Mix carefully using a one milliliter pipette. Once the solution is well mixed, add 500 microliters of the mixture into each well of the 24-well plate.Place the 24-well plate in a 37 degree Celsius incubator. After 30 minutes, take out the plate and add 500 microliters of pre-warmed culture medium on top of the gel. Prior to imaging, turn on the live cell units of the phase contrast and confocal microscopes.To capture dividing cells, place the 24-well plate containing the cells embedded in collagen matrices into the live cell unit of the fluorescence microscope. Using the 10X objective, find the bottom of the plate and then move it up until the microscope focuses at about 500 micrometers from the bottom of the plate. Move the stage around to find cells in different stages of mitosis and select multiple positions for imaging.Set up the timelapse experiment with two-minute intervals. Next, to image the collagen network, configure the confocal microscope to capture only reflected light from the 488 nanometer laser. Then place the plate containing cells in collagen matrices into the live cell unit of this microscope.Find the bottom of the plate and then move the objective lens up until it focuses at about 100 micrometers from the bottom of the plate. Move the stage around to find cells and select multiple positions for imaging. Set up the timelapse experiment with five-minute intervals.Fluorescent images of a dividing H2B-mCherry expressing MDA-MB-231 cell embedded in collagen matrix are shown here. Different phases of mitosis are readily observed as expected. The distribution of collagen fibers in white visualized simultaneously using confocal reflection microscopy changes very little during the course of mitosis.Cell synchronized G2-M phase and embedded in a collagen matrix complete cell division in the first two hours while control asynchronous cells divide randomly. Quantification of the distribution of collagen fibers during interphase and mitotic phases suggest that matrix deformation is minimal during cell division and higher in interphase cells. Once mastered, this technique can be completed in seven days starting from generating the stable cell line to analyzing the video cell division in a 3D matrix.After watching this video, you hopefully have a good understanding of how to synchronize and monitor mammalian cell division in 3D matrices using live cell imaging and how to determine matrix deformation using confocal reflection microscopy and quantitative imaging analysis techniques.

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