Name: anaerobic growth and maintenance of mammalian cell lines
Uploaded: 2018-07-21T15:00:00
Description: Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

The authors thank the Midwestern University Office of Research and Sponsored Programs for their support.

1. Prepare Media for Anaerobic Culture of Various Mammalian Cell Lines
<ol>
	<li>Make the complete PS-74656 medium for an anoxic cell culture25.
	<ol>
		<li>Make the sterile nitrite stock solution (5 M; 100x) by dissolving 17.25 g of nitrite in 50 mL of distilled deionized water, then filter to sterilize it.</li>
		<li>Add 0.5 mL of the nitrite stock solution per 50 mL of low glucose DMEM (1 g/L of glucose) with 110 mg/L of L-glutamine, 584 mg/L of sodium pyruvate, and 10% fetal...

<ol>
	<li>Cummins, J., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+tumours:+causative+agents+or+opportunistic+inhabitants?.">Bacteria and tumours: causative agents or opportunistic inhabitants?.</a> Infectious Agents and Cancer. 8 (1), 11 (2013).</li><li>Liao, J., 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=Enhanced+efficiency+of+generating+induced+pluripotent+stem+(iPS)+cells+from+human+somatic+cells+by+a+combination+of+six+transcription+factors.">Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.</a> Cell Research. 18 (5), 600-603 (2008).</li><li>Park, I. H., Lerou, P. H., Zhao, R., Huo, H., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nature Protocols. 3 (7), 1180-1186 (2008).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1:+mediator+of+physiological+and+pathophysiological+responses+to+hypoxia.">HIF-1: mediator of physiological and pathophysiological responses to hypoxia.</a> Journal of Applied Physiology. 88 (4), 1474-1480 (2000).</li><li>Biedermann, L., Rogler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+microbiota:+its+role+in+health+and+disease.">The intestinal microbiota: its role in health and disease.</a> European Journal of Pediatrics. 174 (2), 151-167 (2015).</li><li>Cui, X. Y., 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=Hypoxia+influences+stem+cell-like+properties+in+multidrug+resistant+K562+leukemic+cells.">Hypoxia influences stem cell-like properties in multidrug resistant K562 leukemic cells.</a> Blood Cells, Molecules, and Diseases. 51 (3), 177-184 (2013).</li><li>Richter, A., Sanford, K. K., Evans, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Oxygen+and+Culture+Media+on+Plating+Efficiency+of+Some+Mammalian+Tissue+Cells.">Influence of Oxygen and Culture Media on Plating Efficiency of Some Mammalian Tissue Cells.</a> JNCI: Journal of the National Cancer Institute. 49 (6), 1705-1712 (1972).</li><li>Packer, L., Fuehr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+concentration+extends+the+lifespan+of+cultured+human+diploid+cells.">Low oxygen concentration extends the lifespan of cultured human diploid cells.</a> Nature. 267, 423 (1977).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer.">On the origin of cancer.</a> Science. 123 (3191), 309-314 (1956).</li><li>D&#246;me, B., Hendrix, M. J. C., Paku, S., T&#243;v&#225;ri, J., T&#237;m&#225;r, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+Vascularization+Mechanisms+in+Cancer.">Alternative Vascularization Mechanisms in Cancer.</a> The American Journal of Pathology. 170 (1), 1-15 (2007).</li><li>Jewell, U. R., 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=Induction+of+HIF-1&#945;+in+response+to+hypoxia+is+instantaneous.">Induction of HIF-1&#945; in response to hypoxia is instantaneous.</a> The FASEB Journal. 15 (7), 1312-1314 (2001).</li><li>Lee, J. W., Bae, S. H., Jeong, J. W., Kim, S. H., Kim, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+(HIF-1)alpha:+its+protein+stability+and+biological+functions.">Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions.</a> Experimental &amp; Molecular Medicine. 36 (1), 1-12 (2004).</li><li>Pagoulatos, D., Pharmakakis, N., Lakoumentas, J., Assimakopoulou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etaypoxia-inducible+factor-1alpha,+von+Hippel-Lindau+protein,+and+heat+shock+protein+expression+in+ophthalmic+pterygium+and+normal+conjunctiva.">Etaypoxia-inducible factor-1alpha, von Hippel-Lindau protein, and heat shock protein expression in ophthalmic pterygium and normal conjunctiva.</a> Molecular Vision. 20, 441-457 (2014).</li><li>Park, T. J., 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=Fructose-driven+glycolysis+supports+anoxia+resistance+in+the+naked+mole-rat.">Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat.</a> Science. 356 (6335), 307 (2017).</li><li>Johnston, I. A., Bernard, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+the+Ethanol+Pathway+in+Carp+Following+Exposure+to+Anoxia.">Utilization of the Ethanol Pathway in Carp Following Exposure to Anoxia.</a> Journal of Experimental Biology. 104 (1), 73 (1983).</li><li>Takahashi, E., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+respiration+sustains+mitochondrial+membrane+potential+in+a+prolyl+hydroxylase+pathway-activated+cancer+cell+line+in+a+hypoxic+microenvironment.">Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.</a> American Journal of Physiology-Cell Physiology. 306 (4), C334-C342 (2014).</li><li>Mandziuk, 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=Effect+of+hypoxia+on+toxicity+induced+by+anticancer+agents+in+cardiomyocyte+culture.">Effect of hypoxia on toxicity induced by anticancer agents in cardiomyocyte culture.</a> Medycyna Weterynaryjna. 70 (5), 287-291 (2014).</li><li>Arai, H., Kodama, T., Igarashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+nirQOP+genes+in+energy+conservation+during+anaerobic+growth+of+Pseudomonas+aeruginosa.">The role of the nirQOP genes in energy conservation during anaerobic growth of Pseudomonas aeruginosa.</a> Bioscience, Biotechnology, and Biochemistry. 62 (10), 1995-1999 (1998).</li><li>M&#252;ller, 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=Biochemistry+and+Evolution+of+Anaerobic+Energy+Metabolism+in+Eukaryotes.">Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes.</a> Microbiology and Molecular Biology Reviews. 76 (2), 444-495 (2012).</li><li>Gupta, K. J., Igamberdiev, A. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anoxic+plant+mitochondrion+as+a+nitrite:+NO+reductase.">The anoxic plant mitochondrion as a nitrite: NO reductase.</a> Mitochondrion. 11 (4), 537-543 (2011).</li><li>Gupta, K. J., Igamberdiev, A. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Nitrogen+Species+in+Mitochondria+and+Their+Implications+in+Plant+Energy+Status+and+Hypoxic+Stress+Tolerance.">Reactive Nitrogen Species in Mitochondria and Their Implications in Plant Energy Status and Hypoxic Stress Tolerance.</a> Frontiers in Plant Science. 7 (369), (2016).</li><li>Kozlov, A. V., Staniek, K., Nohl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrite+reductase+activity+is+a+novel+function+of+mammalian+mitochondria.">Nitrite reductase activity is a novel function of mammalian mitochondria.</a> FEBS letters. 454 (1-2), 127-130 (1999).</li><li>Nohl, H., Staniek, K., Kozlov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+existence+and+significance+of+a+mitochondrial+nitrite+reductase.">The existence and significance of a mitochondrial nitrite reductase.</a> Redox Report. 10 (6), 281-286 (2005).</li><li>Lawson, J. C., Blatch, G. L., Edkins, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+breast+cancer+and+metastasis.">Cancer stem cells in breast cancer and metastasis.</a> Breast Cancer Research and Treatment. 118 (2), 241-254 (2009).</li><li>Plotkin, B. J., 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+method+for+the+long-term+cultivation+of+mammalian+cells+in+the+absence+of+oxygen:+Characterization+of+cell+replication,+hypoxia-inducible+factor+expression+and+reactive+oxygen+species+production.">A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.</a> Tissue and Cell. 50 (Supplement C), 59-68 (2018).</li><li>Sarmento, 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=Cell-based+in+vitro+models+for+predicting+drug+permeability.">Cell-based in vitro models for predicting drug permeability.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 8 (5), 607-621 (2012).</li><li>Collins, S. J., Gallo, R. C., Gallagher, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+growth+and+differentiation+of+human+myeloid+leukaemic+cells+in+suspension+culture.">Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture.</a> Nature. 270 (5635), 347-349 (1977).</li><li>Plotkin, B. J., 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+method+for+the+long-term+cultivation+of+mammalian+cells+in+the+absence+of+oxygen:+Characterization+of+cell+replication,+hypoxia-inducible+factor+expression+and+reactive+oxygen+species+production.">A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.</a> Tissue and Cell. 50, 59-68 (2018).</li></ol>

This method represents first-time mammalian cells that were cultured for extended periods of time in the absence of oxygen. Based on current observations, the anoxic growth capability via a non-fermentative pathway appears to be universal amongst mammalian cells lines28, where the anaerobic growth resulted in phenotype divergence. This was observed for all cell lines tested. With the anaerobic cultivation, increasing proportions of the cells became rounded, developed a suspension-like pop...

Cells from solid tumors, stem cell niches, and those lining the mucosal surfaces exist in environments that experience reduced oxygen levels, including anoxia1,2,3,4.&#160;In normal physiologic states, oxygenation varies beyond that of hypoxia to anoxia (the complete absence of oxygen)5,6.The realization that atmospheric oxygen adversely affects mammalian cell replication and that in vitro cell growth can be optimized under depleted oxygen conditions was reported in the early 1970s.&#160;Richter et al.7 showed that 1&#8211;3% of oxygen levels (hypoxia) enhanced plating efficiency as compared to atmospheric oxygen (20%). The human diploid cell lifespan is also extended in the hypoxic culture conditions8.
In vivo, hypoxic conditions occur when oxygen stores are depleted (e.g.,&#160;during intense exercise), wherein the ATP production is switched in the skeletal muscle from aerobic respiration to fermentation (anaerobic respiration) with the end-product of lactic acid9. Pathologically, in cancerous tumors, the interior of the tumor mass is hypoxic to anoxic due to poor vascularization10. The effect of the limited perfusion on tumor interiors is independently validated by tumor interiors colonized by obligate anaerobes1. Mechanistically, tumor cell survival in hypoxia is thought to be solely dependent on the expression of the hypoxia-inducible factor 1-alpha gene (HIF1-alpha), which is the initial spontaneous response to hypoxia4,11,12. HIF1-alpha is induced under hypoxic conditions by heat shock proteins that bind the HIF1-alpha promoter and upregulate the gene transcription12. These heat shock proteins are believed to aid in the induction of the various phenotypes seen in the tumor hypoxic microenvironment. These phenotypes exhibit an increased expression of the cell membrane glucose transporters and the rate of glycolysis (the Warburg effect)13. The result is a switch from mitochondrial oxidative phosphorylation to lactate fermentation.
Anoxic survival can also utilize alternatives to glucose to support the survival phenomenon14,15. The best studied mammalian example is the mole rat, which can survive for nearly 20 min without oxygen through a fructose-driven glycolytic fermentation pathway14. An alternative adaptation occurs in certain fish (e.g., carp [Carassius sp.], which can survive for significantly longer time periods using glycolysis with ethanol as the terminal by-product)15. In both cases, fermentation drives the metabolism enabling the survival in the absence of oxygen. The current hypothesis for anoxic survival is that so long as HIF1-alpha is activated during hypoxia, mitochondrial respiration, without the need for oxygen, occurs under anaerobic conditions16. Furthermore, it is postulated that the use of a fermentative pathway for hypoxic/anoxic survival enhances tumor survival since the cells avoid oxidative stress which could prove to be detrimental to the cell survival17. This postulate is supported by a recent study which shows that in cardiomyocytes, hypoxia reduces the oxidative stress placed on the tumor cell17.
To date, the essential nature of a fermentative pathway for anoxic mammalian cell survival has been ingrained in the literature, due, in large part, to an inability to culture mammalian cells in the complete absence of oxygen for more than 3 days. However, an alternative to glycolysis for the anaerobic survival occurs in bacteria. In certain bacteria, nitrogen or sulfate (among other compounds) can serve as terminal electron acceptors for the cytochrome oxidase system in the absence of oxygen18. Although the bacterial and eukaryotic evolution occurred in parallel since diverging from the last universal common ancestor, it is estimated that mitochondria were providing energy to cells for 1.542 million years before the oxygenation of the oceans19. Since researchers have shown that the isolated mitochondria can produce ATP in the absence of oxygen, with nitrite as the terminal electron acceptor, it is reasonable to assume that cells could function for periods of time longer than 3 days in the absence of oxygen20,21,22,23. The methodology described in this study has a utility for the anaerobic mammalian cell growth of numerous cell lines.

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="709">
	<tbody>
		<tr height="147">
			<td height="147" style="height:147px;width:255px;">Whitney A35 anaerobic chamber</td>
			<td style="width:149px;">Don Whitley Scientific&#160;</td>
			<td style="width:139px;">Microbiology International</td>
			<td style="width:167px;">The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CO2 incubator</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">3531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue Culture Hood</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:139px;">DO55735</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">pipet aid</td>
			<td style="width:149px;">Drummond</td>
			<td style="width:139px;">4-000-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile transfer pipets</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-358867</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50cc sterile conical centrifuge tubes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">451-PG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">vaccum jar</td>
			<td style="width:149px;">Nalgene 111410862889</td>
			<td style="width:139px;">BTA-Mall 5311-0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM high glucose (4.5 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM low glucose (1 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-014-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FBS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">1500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HBSS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypsin</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">gentamicin</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:139px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile pipets</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229210B, 229205B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75 or T150)</td>
			<td style="width:149px;">Santa Cruz</td>
			<td>sc-200263, sc-200264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">24 well tissue culture treated dishes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">667124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">glad ziplock sandwich bags</td>
			<td style="width:149px;">Ziploc</td>
			<td style="width:139px;">Costco</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">inverted phase microscope (10, 20 40x objectives with camera mont)</td>
			<td style="width:149px;">Nikon Eclipse</td>
			<td style="width:139px;">TS100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypan blue</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">hemocytometer</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Countess Automated Cell Counter</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:139px;">AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Rainin microtiter pipets</td>
			<td style="width:149px;">Mettler Toledo</td>
			<td style="width:139px;">Rainin Classic Pipette PR-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">microtiter tips</td>
			<td style="width:149px;">Santa Cruz Biotechnology</td>
			<td style="width:139px;">sc-213233 &#38; sc-201717&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">test tube rack (50cc tubes)</td>
			<td style="width:149px;">The Lab Depot</td>
			<td>HS29050A</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:255px;">sodium nitrite</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:139px;">https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en&#38;region=US</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">clear boxes with lids</td>
			<td style="width:149px;">Rosti Mepal</td>
			<td style="width:139px;">Rubber maid</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">paper towels</td>
			<td style="width:149px;">In House</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">cell scraper</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:149px;">In house prepared</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Ethanol</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">microcentrifuge tubes sterile</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-200273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">biohazard bags with holder (desktop)</td>
			<td style="width:149px;">Heathrow Scientific</td>
			<td style="width:139px;">HS10320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nitrile Gloves</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">89428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">oxygen electrode</td>
			<td style="width:149px;">eDAQ</td>
			<td style="width:139px;">EPO354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH meter</td>
			<td style="width:149px;">Jenway</td>
			<td style="width:139px;">3510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH paper/ pHydrion</td>
			<td style="width:149px;">Sigma Aldich</td>
			<td>Z111813</td>
			<td></td>
		</tr>
	</tbody>
</table>

anaerobic growth and maintenance of mammalian cell lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

The strength of this protocol lies in its support of the longevity and the growth of multiple cell lines and in the recognition that there are a profound alteration and divergence in the cell morphology25. The most critical element of this study is the transfer and maintenance of the cells under strict anaerobiosis. This requires an anaerobic chamber organized to maximize the protocol (Figure 1) and the assurance that the cells removed...

Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

This method can be used to characterize the physiology of anoxic cells and help answer key questions in host pathogen interactions, stem cell differentiation and cancer cell metastasis and therapeutic susceptibility. This is the first described protocol that enables the long-term anoxic cultivation of mammalian cells. The implications of this technique extend toward targeted, personalized cancer therapy and rejuvenative therapy because, due to oxygen toxicity, multiple cell types prefer low-oxygen environments for growth.Though this method can provide insight into cell anaerobic respiration and metabolism, it can also be applied to other systems such as stem cell cultivation. Generally, individuals new to this method will struggle because extreme care must be exercised to prevent oxygen contamination. Physical demonstration of this method is critical, as the care needed to exclude oxygen contamination is not intuitive.To begin, add 0.5 milliliters of nitrite stock solution to low glucose DMEM containing L-glutamine, sodium pyruvate and fetal bovine serum. Then place 20 milliliters of the medium in a sterile 50 milliliter conical centrifuge tube. Next, degas the medium by placing the tube in a vacuum bell jar at a 45 degree angle.Use a vacuum pump to apply a vacuum at room temperature. Maintain the vacuum for 24 to 48 hours, until bubbles are no longer observed. Remove the tube from the bell jar and close the cap immediately to minimize introduction of air to the medium.Then place the tube for 24 hours in a prepared anaerobic chamber and loosen the cap. While the tube is in the chamber, transfer 100 microliters of the medium to a sterile, degassed microcentrifuge tube. Then use an oxygen probe to test the tube for dissolved oxygen.First, grow cells in a T150 flask to 90%confluence. Remove the medium from the flask via aspiration and wash the flask with 10 milliliters of HBSS. Then, replace the removed HBSS with five milliliters of trypsin and agitate the flask until the cells detach from the plastic.Aspirate most of the trypsin from the flask and tap the flask to dislodge any adhered cells. Then add 10 to 15 milliliters of high glucose DMEM to inactivate the trypsin. Use a 25 milliliter pipette to triturate the cells until all of the cell clumps are disrupted.Then, using a standard hemocytometer, count the cells and determine the viability. Suspend the cells in high glucose DMEM and add one milliliter of the suspension to the wells of a 24-well tissue culture plate. Then, incubate the cells at 37 degrees Celsius in normoxic conditions for 24 hours.After the 24 hour incubation period, transfer the plate to the anerobic chamber. Then, immediately change the medium to antibiotic-free, degassed PS-74656 medium. Place the plate in a container with a damp towel and loosely close it.After the second 24-hour incubation period, continue with further incubation periods, as outlined in the text protocol. Monitor the anaerobic medium for color change over time. When the medium changes from magenta to red, remove the runagate cells via aspiration.Transfer the aspirated spent media to a prepared microfuge tube. Close the tube and centrifuge it. Replace the aspirated media in the wells of the plate with new degassed PS-74656 medium.Next, place the microfuge tube with the pelleted cells in the anaerobic chamber before opening it. Aspirate the spent medium and replace it with new degassed PS-74656 medium to a total of one milliliter in the tube. Next, transfer the cells from the microfuge tube to a well in the tissue culture plate.For microscopy, while in the anaerobic chamber, place the plate in a resealable bag flushed with anaerobic gas mixture. Then close the bag. Seal the bag completely and remove it from the chamber.Stretch the bag over the plate and use an inverted phase contrast microscope to examine the cells. Return the plate in the sealed bag to the chamber and incubate the plate. Finally, separately analyze the runagate cells in suspension, according to the text protocol.Using this protocol, established cell lines were successfully cultivated in an anaerobic environment. Even after an extended incubation of several weeks, some cells remain tethered, while the majority displayed rounded runagate morphology. Water droplets can create microscopy artifacts that are frequently mistaken for cells.Final cell viability for long-term cultivation was dependent on the confluence to which the wells or flask were initially seeded. HeLa 229 cell growth was optimized at 80%initial seeding confluence. Vero cell growth, however, was optimized at 60%initial seeding confluence.Once mastered, this technique can become a routine procedure for maintaining cells anoxically. While attempting this procedure, it's important to remember to pre-prepare medium so that it's properly degassed and to degas all other materials used, such as microcentrifuge tubes and pipette tips. Following this procedure, other methods, like drug cytotoxicity testing and stem cell cultivation, can be performed in order to answer additional questions, like anaerobic cancer cell drug sensitivity and anoxic cytokine expression.After it's development, this technique paved the way for researchers in the field of host pathogen interactions, cancer cell chemotherapy and stem cell propagation to explore natural changes in cell physiology that can occur in vivo. After watching this video, you should have a good understanding of how to culture mammalian cells for long periods of time in the absence of oxygen. Don't forget that working with cancer cells can be extremely hazardous and precautions, such as handling of bloodborne pathogens, should always be taken while performing this procedure.

anaerobic-growth-and-maintenance-of-mammalian-cell-lines

Research

JoVE Journal

Biology

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...