Name: evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells
Uploaded: 2020-06-13T14:45:00
Description: Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Dit onderzoek werd ondersteund door de "Vaststelling van meetnormen voor chemie en straling", subsidienummer KRISS-2020-GP2020-0003 en "Ontwikkeling van meetnormen en technologie voor biomaterialen en medische convergentie", subsidienummer KRISS-2020-GP2020-0004-programma's, gefinancierd door het Korea Research Institute of Standards and Science. Dit onderzoek werd ook ondersteund door het Ministerie van Wetenschap en ICT (MSIT), National Research Foundation of Korea (NRF-2019M3A9F3065868), het Ministerie van Volksgezondheid en Welzijn (MOHW), het Korea Health Industry Development Institute (KHIDI, HI20C0558), het Ministerie van Handel, Industrie & Energie (MOTIE) en korea Evaluation Institute of Industrial Technology (KEIT, 20009350). ORCID-ID (Hee Min Yoo: 0000-0002-5951-2137; Dukjin Kang: 0000-0002-5924-9674; Seil Kim: 0000-0003-3465-7118; Joo-Eun Lee: 0000-0002-2495-1439; Jina Lee: 0000-0002-3661-3701). We danken Chang Woo Park voor hulp bij experimenten.

1. Bacteriële celculturen en bereiding van Lactobacillus celvrij supernatans (LCFS)
OPMERKING: De stappen 1.2 – 1.9 worden uitgevoerd in een anaerobe kamer.
<ol><li>Bereid een MRS-agarplaat en bouillon met L-cysteïne en steriliseer door autoclaaf.</li>
	<li>Voorbevroom de MRS-agarplaat in de anaerobe H2-kamer bij 37 °C met 20 ppm zuurstof.</li>
	<li>Ontdooi lactobacillus bacteriebestand en ent de agarplaat met de bacteriecultuur (figuur ...

<ol>
	<li>Bron, P. A., Van Baarlen, P., Kleerebezem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+molecular+insights+into+the+interaction+between+probiotics+and+the+host+intestinal+mucosa.">Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa.</a> Nature Reviews Microbiology. 10 (1), 66-78 (2012).</li><li>Ruiz, L., Delgado, S., Ruas-Madiedo, P., S&#225;nchez, B., Margolles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifidobacteria+and+their+molecular+communication+with+the+immune+system.">Bifidobacteria and their molecular communication with the immune system.</a> Frontiers in Microbiology. 8, 1-9 (2017).</li><li>Sanders, M. E., Merenstein, D. J., Reid, G., Gibson, G. R., Rastall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+and+prebiotics+in+intestinal+health+and+disease:+from+biology+to+the+clinic.">Probiotics and prebiotics in intestinal health and disease: from biology to the clinic.</a> Nature Reviews Gastroenterology and Hepatology. 16 (10), 605-616 (2019).</li><li>Pandey, K. R., Naik, S. R., Vakil, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics,+prebiotics+and+synbiotics-+a+review.">Probiotics, prebiotics and synbiotics- a review.</a> Journal of Food Science and Technology. 52 (12), 7577-7587 (2015).</li><li>Harish, K., Varghese, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+in+humans-evidence+based+review.">Probiotics in humans-evidence based review.</a> Calicut Medical Journal. 4 (4), 3 (2006).</li><li>Routy, B., 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=The+gut+microbiota+influences+anticancer+immunosurveillance+and+general+health.">The gut microbiota influences anticancer immunosurveillance and general health.</a> Nature Reviews Clinical Oncology. 15 (6), 382-396 (2018).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+of+the+cell-based+data-harvesting+efforts+that+drive+the+integration+of+cell+biology.">Most of the cell-based data-harvesting efforts that drive the integration of cell biology.</a> Nature Reviews Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+Experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: Experimental models of mammalian biology and disease.</a> Nature Reviews Molecular Cell Biology. 15 (10), 647-664 (2014).</li><li>Jong, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Seminars in Cancer Biology. 15 (5), 365-377 (2005).</li><li>Zanoni, M., 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=3D+tumor+spheroid+models+for+in+vitro+therapeutic+screening:+a+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.</a> Scientific Reports. 6, 19103 (2016).</li><li>Anton, D., Burckel, H., Josset, E., Noel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+breakthrough+in+vivo.">Three-dimensional cell culture: A breakthrough in vivo.</a> International Journal of Molecular Sciences. 16 (3), 5517-5527 (2015).</li><li>Lee, J. E., 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=Characterization+of+the+Anti-Cancer+Activity+of+the+Probiotic+Bacterium+Lactobacillus+fermentum+Using+2D+vs.+3D+Culture+in+Colorectal+Cancer+Cells.">Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus fermentum Using 2D vs. 3D Culture in Colorectal Cancer Cells.</a> Biomolecules. 9 (10), 557 (2019).</li><li>Koledova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture:+An+introduction.">3D cell culture: An introduction.</a> Methods in Molecular Biology. 1612, (2017).</li><li>Kapa&#322;czy&#324;ska, M., 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=2D+and+3D+cell+cultures+-+a+comparison+of+different+types+of+cancer+cell+cultures.">2D and 3D cell cultures - a comparison of different types of cancer cell cultures.</a> Archives of Medical Science. 14 (4), 910-919 (2018).</li><li>Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+cell+culture+models+in+drug+discovery+and+drug+repositioning.">Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.</a> Frontiers in Pharmacology. 9, 1-14 (2018).</li><li>Breslin, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+The+missing+link+in+drug+discovery.">Three-dimensional cell culture: The missing link in drug discovery.</a> Drug Discovery Today. 18 (5-6), 240-249 (2013).</li><li>Mazzocchi, A. R., Rajan, S. A. P., Votanopoulos, K. I., Hall, A. R., Skardal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+patient-derived+3D+mesothelioma+tumor+organoids+facilitate+patient-centric+therapeutic+screening.">In vitro patient-derived 3D mesothelioma tumor organoids facilitate patient-centric therapeutic screening.</a> Scientific Reports. 8, 2886 (2018).</li><li>Lv, D., Hu, Z., Lu, L., Lu, H., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+powerful+tool+in+tumor+research+and+drug+discovery.">Three-dimensional cell culture: A powerful tool in tumor research and drug discovery.</a> Oncology Letters. 14 (6), 6999-7010 (2017).</li><li>Thirumala, S., Gimble, J., Devireddy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+Based+Thermally+Reversible+Hydrogel+System+for+Tissue+Engineering+Applications.">Methylcellulose Based Thermally Reversible Hydrogel System for Tissue Engineering Applications.</a> Cells. 2 (3), 460-475 (2013).</li><li>Chandrashekran, A., 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=Methylcellulose+as+a+scaffold+in+the+culture+of+liver-organoids+for+the+potential+of+treating+acute+liver+failure.">Methylcellulose as a scaffold in the culture of liver-organoids for the potential of treating acute liver failure.</a> Cell and Gene Therapy Insights. 4 (11), 1087-1103 (2018).</li><li>Lee, W., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+patterned+stem+cell+differentiation+using+thermo-responsive+methylcellulose+hydrogel+molds.">3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds.</a> Scientific Reports. 6, 1-11 (2016).</li><li>Fan, H., Demirci, U., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+organoid+models:+Leaping+forward+in+cancer+research.">Emerging organoid models: Leaping forward in cancer research.</a> Journal of Hematology and Oncology. 12 (1), 1-10 (2019).</li><li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature Reviews Cancer. 18 (7), 407-418 (2018).</li><li>Liou, C. S., 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=A+Metabolic+Pathway+for+Activation+of+Dietary+Glucosinolates+by+a+Human+Gut+Symbiont.">A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.</a> Cell. 180 (4), 717-728 (2020).</li><li>Sherwin, E., Bordenstein, S. R., Quinn, J. L., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+and+the+social+brain.">Microbiota and the social brain.</a> Science. 366 (6465), 2016 (2019).</li><li>Honda, K., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiota+in+adaptive+immune+homeostasis+and+disease.">The microbiota in adaptive immune homeostasis and disease.</a> Nature. 535 (7610), 75-84 (2016).</li><li>B&#225;rcena, C., 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=Healthspan+and+lifespan+extension+by+fecal+microbiota+transplantation+into+progeroid+mice.">Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.</a> Nature Medicine. 25 (8), 1234-1242 (2019).</li><li>Michalovich, D., 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=Obesity+and+disease+severity+magnify+disturbed+microbiome-immune+interactions+in+asthma+patients.">Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients.</a> Nature Communications. 10, 5711 (2019).</li><li>Ansaldo, E., 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=Akkermansia+muciniphila+induces+intestinal+adaptive+immune+responses+during+homeostasis.">Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis.</a> Science. 364 (6446), 1179-1184 (2019).</li></ol>

Het weefselmicromilieu, met inbegrip van naburige cellen en de extracellulaire matrix (ECM), is fundamenteel voor weefselgeneratie en cruciaal voor de controle van celgroei en weefselontwikkeling13. 2D-culturen hebben echter verschillende nadelen, zoals de verstoring van cellulaire interacties, evenals veranderingen in de celmorfologie, extracellulaire omgevingen en de benadering van divisie14. 3D-celkweeksystemen zijn grondig bestudeerd om in vivo effecten beter te reprodu...

Probiotica zijn de meest voordelige micro-organismen in de darm die de immuunhomeostase verbeteren en het energiemetabolisme hosten1. Probiotica van Lactobacillus en Bifidobacterium zijn de meest geavanceerde in zijn soort gevonden in de darm2,3. Eerdere onderzoeken hebben aangetoond dat Lactobacillus remmende en antiproliferatieve effecten heeft op verschillende kankers, waaronder colorectale kanker4. Bovendien voorkomen probiotica inflammatoire darmziekten, de ziekte van Crohn en colitis ulcerosa5,6. Echter, de meeste studies met probiotica werden uitgevoerd in tweedimensionale (2D) monolagen die worden gekweekt op vaste oppervlakken.
Kunstmatige kweeksystemen missen omgevingskenmerken, wat niet natuurlijk is voor kankercellen. Om deze beperking te overwinnen, zijn driedimensionale (3D) kweeksystemen ontwikkeld7,8. Kankercellen in 3D vertonen verbeteringen in termen van biologische basismechanismen, zoals cel levensvatbaarheid, proliferatie, morfologie, celcelcommunicatie, medicijngevoeligheid en in vivo relevantie9,10. Bovendien zijn sferoïden gemaakt van meercellige soorten colorectale kanker en zijn ze afhankelijk van cel-celinteracties en de extracellulaire matrix (ECM)11. Onze vorige studie heeft gemeld dat probiotische celvrije supernatant (CVS) geproduceerd met behulp van Lactobacillus fermentum toonde anti-kanker effecten op 3D culturen van colorectale kanker (CRC) cellen12. We stelden voor dat CVS een geschikte alternatieve strategie is voor het testen van probiotische effecten op 3D-sferoïden12.
Hier presenteren we een aanpak die geschikt is voor meercellige soorten 3D-colorectale kanker voor de analyse van therapeutische effecten van probiotische celvrije supernatant (CVS) op verschillende 3D-colorectale kanker mimicry systemen. Deze methode biedt een middel voor de analyse van gerelateerde probiotische en anti-kanker effecten in vitro.

<table>
	<tbody>
		<tr>
			<td> 
			10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 &#181;l</td>
			<td>Biorad</td>
			<td>4561036</td>
			<td>Pkg of 10</td>
		</tr>
		<tr>
			<td>Applied Biosystems MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>100 covers</td>
		</tr>
		<tr>
			<td>10x transfer buffer</td>
			<td>Intron</td>
			<td>IBS-BT031A</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>10X Tris-Glycine (W/SDS)</td>
			<td>Intron</td>
			<td>IBS-BT014</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case</td>
			<td>Corning</td>
			<td>SCT-200-C</td>
			<td>500 Tubes/Pack, 10 Packs/Case</td>
		</tr>
		<tr>
			<td>BD Difco Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>BD Difco Lactobacilli MRS Broth</td>
			<td>BD</td>
			<td>DF0881-17-5</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell viability assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>100&#956;l/assay in 96-well plates</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td>vial of 20 tablets</td>
		</tr>
		<tr>
			<td>Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 &#177; 0.1</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>EMD Millipore Immobilon-P PVDF Transfer Membranes</td>
			<td>fisher Scientific</td>
			<td>IPVH00010</td>
			<td>26.5cm x 3.75m roll; Pore Size: 0.45um</td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>25/Pack, 500/Case</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit, 25 x 20 &#181;l rxns #1708890</td>
			<td>Biorad</td>
			<td>1708890</td>
			<td>25 x 20 &#181;L rxns</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Biorad</td>
			<td>1725121</td>
			<td>5 x 1 mL</td>
		</tr>
		<tr>
			<td>Lactobacillus fermentum</td>
			<td>Korean Collection for Type Cultures</td>
			<td>KCTC 3112</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6852-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Methyl Cellulose (3500-5600mPa&#183;s, 2% in Water at 20&#176;C)</td>
			<td>TCI</td>
			<td>M0185</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td>20 plates</td>
		</tr>
		<tr>
			<td>Millex-GS Syringe Filter Unit, 0.22 &#181;m, mixed cellulose esters, 33 mm, ethylene oxide sterilized</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>250</td>
		</tr>
		<tr>
			<td>PE Annexin V Apoptosis Detection Kit with 7-AAD</td>
			<td>Biolegend</td>
			<td>640934</td>
			<td>100 tests</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Introgen</td>
			<td>P1304MP</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>89901</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>250</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Name of Materials/Equipment/Software</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>anti - p-I&#954;B&#945; (B-9)</td>
			<td>Santa cruze</td>
			<td>sc-8404</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-BclxL (H-5)</td>
			<td>Santa cruze</td>
			<td>sc-8392</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-PARP 1 (C2-10)</td>
			<td>Santa cruze</td>
			<td>sc-53643</td>
			<td>50 &#181;l ascites</td>
		</tr>
		<tr>
			<td>anti-&#946;-actin (C4)</td>
			<td>Santa cruze</td>
			<td>sc-47778</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BD FACSVerse</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Microplate Reader</td>
			<td>BioT</td>
			<td>S1LFA</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo fisher</td>
			<td>HERAcell 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 15 ml</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 50 ml</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile</td>
			<td>Costar</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermofisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EnSpire Multimode Reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Enspire 2300</td>
		</tr>
		<tr>
			<td>Eppendorf Research Plus Multi Channel Pipette, 8-channel</td>
			<td>Eppendorf</td>
			<td>3122000051</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>TreeStar</td>
			<td></td>
			<td>Ashland, OR, USA</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>115-035-062</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>111-035-144</td>
			<td>2.0 mL</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Inc., San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Incubated shaker</td>
			<td>Lab companion</td>
			<td>SIF-6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Gauge Ver. 3.0,</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Optical density (OD)LAMBDA UV/Vis Spectrophotometers</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>SPL microcentrifuge tube 1.5mL</td>
			<td>SPL</td>
			<td>60015</td>
			<td></td>
		</tr>
		<tr>
			<td>SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile</td>
			<td>SPL</td>
			<td>21012</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Vibra-Cell Ultrasonic Liquid Processors</td>
			<td>SONICS-vibra cell</td>
			<td>VC 505</td>
			<td>500 Watt ultrasonic processor</td>
		</tr>
		<tr>
			<td>Vinyl Anaerobic Chamber</td>
			<td>COY LAB PRODUCTS</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells

Dit manuscript beschrijft een protocol om sterfgevallen door kankercellen te evalueren in driedimensionale (3D) sferoïden van meercellige soorten kankercellen met behulp van supernatanten uit lactobacillus fermentumcelcultuur , beschouwd als probioticaculturen. Het gebruik van 3D-culturen om Lactobacillus celvrij supernatant (LCFS) te testen is een betere optie dan testen in 2D-monolagen, vooral omdat L. fermentum antikankereffecten in de darm kan veroorzaken. L. fermentum supernatant bleek verhoogde antiprolifererende effecten te bezitten tegen verschillende colorectale kankercellen (CRC) in 3D-kweekomstandigheden. Interessant is dat deze effecten sterk gerelateerd waren aan het kweekmodel, wat het opmerkelijke vermogen van L. fermentum aantoonde om kankerceldood te induceren. Stabiele sferoïden werden gegenereerd uit verschillende CRC's (colorectale kankercellen) met behulp van het onderstaande protocol. Dit protocol van het genereren van 3D spheroid is tijdbesparend en kosteneffectief. Dit systeem is ontwikkeld om eenvoudig de antikankereffecten van LCFS in meerdere soorten CRC-sferoïden te onderzoeken. Zoals verwacht, crc spheroids behandeld met LCFS sterk geïnduceerde celdood tijdens het experiment en uitgedrukt specifieke apoptosis moleculaire markers zoals geanalyseerd door qRT-PCR, western blotting, en FACS analyse. Daarom is deze methode waardevol voor het onderzoeken van de levensvatbaarheid van cellen en het evalueren van de werkzaamheid van antikankergeneesmiddelen.

We beschrijven het protocol van het verkrijgen van sferoïden uit verschillende colorectale kankercellijnen. Suppletie met methylcellulose was nodig om sferoïden te genereren. We presenteren ook een methode van LCFS voorbereiding en presenteren een model om de correlatie tussen probiotica en colorectale kanker te bestuderen. Sferoïde vormings - en LCFS-voorbereidingsprotocollen worden schematisch geïllustreerd in figuur 1A,B. Zoals weergegeven in figu...

Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Hier methoden worden gepresenteerd om te begrijpen anti-kanker effecten van Lactobacillus celvrije supernatant (LCFS). Colorectale kankercellijnen tonen celsterfte bij behandeling met LCFS in 3D-culturen. Het proces van het genereren van sferoïden kan worden geoptimaliseerd, afhankelijk van de steiger en de gepresenteerde analysemethoden zijn nuttig voor het evalueren van de betrokken signaleringsroutes.

Celdood evalueren met celvrije supernatant van probiotica in driedimensionale sferoïde culturen van colorectale kankercellen

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

Gebruik makend van Functional Genomics Screening om potentieel nieuwe drug targets in Cancer Cell Sferoïde Cultures Identificeer

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Voorbereiding en metabole kwantitatieve analyse van 3-dimensionale sferoïde co culturen van Pancreatic kankercellen en fibroblasten

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Verplaatsen naar boven: Een eenvoudig en flexibel In Vitro driedimensionale invasie Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Volledig menselijke tumor-gebaseerde matrijs in driedimensionele sferoïde invasie assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Beoordeling van de levensvatbaarheid van cellen en de dood in 3D sferoïde culturen van kankercellen

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

Evaluatie van de differentiatie capaciteit van muizen prostaat epitheelcellen met behulp van Organoid cultuur

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Isolatie van stam-achtige cellen uit 3-dimensionale Spheroid-culturen

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Detectie van celvrij DNA in bloedplasmamonsters van kankerpatiënten

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Een driedimensionaal model van sferoïden om stamcellen van darmkanker te bestuderen

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Driedimensionaal in vitro biomimetisch model van neuroblastoom met behulp van op collageen gebaseerde steigers

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...