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 bovine serum (FBS; heat-inactivated). 
		NOTE: The use of FBS concentrations greater than 10% are toxic. Of note, one cancer cell line tested had a low tolerance for media with nitrite (MDA-MB-231, breast cancer cell line) and, thus, was grown in the absence of nitrite. In addition, although PS-74656 medium supported the growth of all cell lines that were tested (n = 9), certain cell lines (e.g., Vero cells) exhibited higher cell concentrations and a higher viability in PS-74656 with high glucose (4.5 g/L) DMEM25.</li>
		<li>Place 20 mL of medium in a sterile 50 mL conical centrifuge tube. Do not tighten the cap; it should be loose.</li>
		<li>De-gas the medium by placing the tube in a vacuum bell jar at an approximately 45&#176; angle, to maximize the medium surface area.</li>
		<li>Apply a vacuum using a vacuum pump, or its equivalent, at room temperature. Maintain the vacuum until bubbles are no longer observed (24&#8211;48 h).</li>
		<li>Remove the tube from the jar and immediately close the cap, sealing the tube to minimize the introduction of air into the medium.</li>
		<li>Place the tube in the commercial anaerobic chamber.</li>
		<li>Loosen the tube cap and allow the atmosphere in the tube to equilibrate with that of the anaerobic gas mixture in the chamber for at least 24 h. 
		NOTE: The medium should remain in the chamber until it is used up. For speeding up the process, flush the tube at least 3x with the anoxic gas mixture using a sterile transfer pipette filled with anoxic gas, which consists of H2, CO2, and N2. </li>
		<li>Degas all tubes (10&#8211;15 min) placed in the anaerobic chamber as described above before sealing the tube and placing in the chamber. Before use, flush it with the anaerobic gas mixture using 1.5&#8211;2 mL transfer pipettes or pipette tips that have been flushed with anoxic gas at least 3x.</li>
		<li>While in the anaerobic chamber, remove 100 &#181;L of the medium to a sterile degassed micro-centrifuge tube and test it for the dissolved oxygen using an oxygen probe within the chamber. 
		NOTE: The oxygen electrode is calibrated per the manufacturer&#39;s protocol prior to use. Readings of the properly degassed and equilibrated anaerobic gas mixture media are 0. 
		CAUTION: If the readings are above 0.3 ppm of oxygen, continue to incubate the media in the anaerobic chamber since more time to equilibrate the medium is required.</li>
	</ol>
	</li>
</ol>
2. Anoxic Cultivation of Various Mammalian Cell Lines
<ol>
	<li>Designated day -1
	<ol>
		<li>Grow cells (37 &#176;C, 5% CO2) in a T150 flask to 90% confluence for the anoxic and normoxic culture control to provide a sufficient number of cells for three 24-well plates (80% confluence). 
		NOTE: For this study, HeLa 229 and Vero cells were used. This protocol was also tested and proved successful for the growth and maintenance of seven other cell lines25.</li>
		<li>Remove the medium from the flask by aspiration and wash it with 10 mL of HBSS.</li>
		<li>Replace the medium with 5 mL of trypsin. Agitate the flask until the cells visually detach from the plastic. Aspirate most of the trypsin, then tap the flask to dislodge the adherent cells. Add 10&#8211;15 mL of high glucose DMEM (4.5 g/L of glucose, 110 mg/L of L-glutamine, 10% FBS, 50 &#181;g/mL of gentamicin) to inactivate the trypsin.</li>
		<li>Triturate the cells using a 25 mL pipette until all cell clumps are disrupted.</li>
		<li>Count the cells and determine the viability using the standard hemocytometer and the trypan blue dye exclusion method or the automated equivalent.</li>
		<li>Suspend the cells in the high glucose DMEM as mentioned above to a cell density of 2.24 x 105 cells/mL.</li>
		<li>Add 1 mL of the cell suspension to the wells of a 24-well tissue culture plate (2.24 x 105 cells/well; 80% confluence). Seed 1 plate for the anoxic culture and another for a control normoxic culture.</li>
		<li>Incubate the cells at 37 &#176;C in normoxic conditions (5% CO2) for 24 h. 
		NOTE: The cells can be plated in various well-sized plates. However, seeding the cells for anoxic growth in flasks runs the risk of the introduction of oxygen into the system, unless care is exercised to completely flush out atmospheric gas.</li>
	</ol>
	</li>
	<li>Designated day 0
	<ol>
		<li>After 24 h of the normoxic incubation, place the plates for the anoxic growth into the anaerobic chamber (a commercial anaerobic chamber; Figure 1).</li>
		<li>Immediately change the medium to antibiotic-free de-gassed PS-74656 medium which has been acclimated for 24 h to the anaerobic chamber. Place the plate in a container with a damp towel and loosely close it to maintain the moisture. 
		NOTE: The plates for normoxic growth are handled identically with the exception that all the treatments are done under atmospheric conditions.</li>
	</ol>
	</li>
	<li>Designated Day 1
	<ol>
		<li>Day 1 of the anaerobic incubation starts after 24 h of the incubation in the commercial anaerobic chamber which contains the standard anaerobic gas mixture of 10% H2, 10% CO2, and 80% N2.</li>
		<li>Incubate the cells for various time periods.</li>
		<li>Monitor the anaerobic medium for the color change over time. 
		NOTE: A color shift from red-orange to magenta signals the time to change the medium. This can take up to 2 weeks to occur. A color shift in the medium from red-orange to yellow indicates the presence of a bacterial contamination.</li>
		<li>When the medium changes from red to magenta, remove the runagate cells by aspiration.</li>
		<li>Place the aspirated spent media with the runagate cells in degassed and anaerobic gas equilibrated microfuge tubes, close the tubes while making sure that the seal is secure, and then centrifuge them (326 x g; at room temperature for 10 min). Immediately replace the aspirated media in the well with 1 mL of new degassed PS-74656 medium.</li>
		<li>Place the microfuge tube with pelleted cells in the anaerobic chamber prior to opening it. Aspirate the spent medium and replace it with fresh degassed PS-74656 medium to a total volume of 1 ml in the microfuge tube.</li>
		<li>Return the cells from the tube to a well in the 24-well tissue culture plate.</li>
	</ol>
	</li>
</ol>
3. Assessment of Phenotypic Cell Differentiation by Microscopy of Anaerobically Cultured Cells
<ol>
	<li>While in the anaerobic chamber, place the plate in a resealable box flushed with the anaerobic gas mixture and close the box. 
	NOTE: The box must contain a damp paper towel to provide moisture and prevent the plate from drying out. For microscopy, sandwich bags work the best since they are thin.
	<ol>
		<li>To observe the cells under the microscope, completely seal the bag and remove it from the chamber.</li>
		<li>Carefully stretch the plastic bag over the plate, examine cells using an inverted phase contrast microscope with the camera attachment and take photomicrographs at various magnifications.</li>
		<li>Return the plate in the sealed bag to the chamber, and continue the incubation as described in step 2.3.3.</li>
		<li>To separately analyze the runagate cells that are in suspension, follow the steps outlined in step 2.3.5. 
		NOTE: These cells can then be used for aerobic testing or can be returned to the anaerobic chamber for the testing of this specific population.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 population, and were able to replicate. The secondary cell type remained attached to the substrate; however, the cell morphology reverted to a spindle (dendritic) shape. These findings point out a common error in analyses and observations with regard to anoxic tissue culture cultivation. It is generally accepted that when mammalian cells grown under normoxic conditions (5% CO2) round up and detach, they are dying. The contrary appears to be correct for cells grown in the absence of oxygen, in which case, cells that have rounded up and detached, are simply a morphologically divergent cell subpopulation. 
The optimization of anoxic growth conditions is cell line dependent. Medium PS-74656 overall supported the growth of all cell lines under normoxic and anoxic growth conditions, with the exception of MDA-MB-231, a breast cancer cell line which preferred growth in the absence of nitrite. An encouraging observation is that even with a 98% die-off of cells per well, the remainder replicates, resulting in a highly adapted population. The critical action is to maintain cultures even when only a few cells are directly observed by microscopy. Thus, care must be exercised to ensure that the detached cells are dead, either by trypan blue dye exclusion or by an alternative cell viability detection method. It should be noted that with such a large decrease in cell numbers, sufficient wells must be seeded to have adequate total cell numbers for subsequent experiments. The expected loss of viable cells is both cell line and seeding confluence dependent; thus, optimal cell numbers, over time, must be determined experimentally and cannot be extrapolated from findings with other cell lines. That said, a good starting point is an 80% confluence, which has the greatest chance of optimal growth. It must be noted that any exposure to oxygen during the anaerobic steps in the procedure, no matter how brief, will compromise the findings due to the reported spontaneous cellular HIF-1-alpha response11,12. 
For this procedure, the most critical step is that of the de-gassing of the medium, tubes, and all other apparatuses and materials used. The introduction of even very low levels of oxygen could stop the transition from the fermentation metabolism to the anaerobic respiration. Alternatively, it could prolong the fermentative metabolism so that any experiments result in the measurement of hypoxia, not anoxia. The use of this novel procedure to measure cytotoxicity and chemotherapeutic activity could help to explain drug failure in the treatment of solid tumors.

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 for either microscopy or other testing are kept in a sealed environment. To date, the range of anoxic survival was 15&#8211;17 days for immortal cancer cell lines and up to 17 days for transformed cell lines (nine cell lines screened for anoxic growth). The levels of viable cells, once these cells were moved to anoxic conditions, declined at least 10%, to a maximum of 90%, before they stabilized. All cell lines reverted to the dual phenotypes (tethered and runagate) with the proportion of cells in the suspension increasing over time. Even after an extended incubation of several weeks, there remained some tethered cells, but the majority of the cells were in the rounded runagate morphology (Figure 2). As reported, these runagate cells are viable and proceeded through their cell cycle. It should be noted that the variables that may need to be adjusted include the glucose levels and the nitrite concentration. That said, from the over 60 different media formulations that were tested for their ability to support both normoxic and anoxic incubation, the PS-74656 medium was able to support the survival and replication of nearly all the cell lines tested.
An essential observation was the differentiation of anoxic cells, regardless of the cell tested, into two distinct phenotypes, tethered and runagate (Figure 2). Analogous to the normoxic monolayer cells, the tethered cells were attached to the tissue culture plastic. However, the attachment was weaker, easier to trypsinize or dislodge by scraping, than the attachment that occurred under the normoxic conditions. In addition, the cell morphology was smaller with more spindle (dendritic) extensions26. The second cellular morphotype observed were cells in suspension (i.e., runagate cells). These cells spontaneously converted into free-floating single cell suspensions in the presence of tissue culture-treated plastic, which were rounded with a smaller cell size and less cytoplasm. They differed from the classical suspension culture cells, which typically require an enzymatic adaptation, grow in aggregates, require non-tissue culture-treated containers, and are larger than their normoxic counterparts27. Over time, the anoxic cell populations could be observed to shift from a predominantly tethered monolayer to that of the runagate cells in suspension. These cells in suspension were viable and replicating; thus, care had to be exercised should the supernatant be removed. Also, of note is that the medium did not require changing over the entire anaerobic incubation period. The medium pH indicator under the anoxic incubation, in contrast to its normoxic control, was also unchanged (i.e., remained red). Should the investigator wish to harvest the runagate cells, or change the medium, the cells can be obtained using anaerobic gas-flushed micro-centrifuge tubes that are closed tightly within the anaerobic chamber. For the cell washing of tethered cells, the procedure must also be performed in the chamber to eliminate any oxygen exposure.
The final cell viability for a long-term cultivation was, to a great extent, dependent on the cell being used and the confluence to which the wells or the flask were initially seeded (Figures 3 and 4). That said, a good starting point is 80%, which had the greatest chance of optimal growth. In addition, a cell line-dependent loss of 10% to 60% of viability can occur in the initial 2&#8211;4 days. However, during the performance of this experiment, the remaining cells cycled through the cell cycle and replicated28. The seeding confluence effect on the longevity of the cell cultivation can be seen in Figures 3 and 4. Both HeLa 229 and Vero cells were seeded in 24-well plates to 40%, 60%, and 80% of initial confluence (100% confluence = 2.8 x 105 cells/well) and then incubated in the absence of oxygen for 17 days. The HeLa 229 cell anoxic growth was optimized at an initial seeding confluence of 80%. In contrast, the Vero cells optimal seeding confluence was 60%. This demonstrates the need to initially optimize the seeding density dependent on the cell line used.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58049/58049fig1.jpg" /> 
Figure 1: Model of anaerobic chamber set-up for culture of mammalian cells. The minimal materials required include tissue culture plates, sterile transfer pipets, biohazard bags, de-gassed media, a tube for the discarding of media or other liquids, and resealable bags for the transfer of the plates to the microscope while maintaining anaerobic conditions.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58049/58049fig2.jpg" /> 
Figure 2: Anoxic HeLa cells morphologic differentiation. (A) This panel shows the phase contrast microscopy of the normoxic HeLa cell controls 4 days post-inoculation, with a 20X magnification. (B) This panel shows the anoxic HeLa cells 17 days post-inoculation (with the unchanged from day 1 PS-74656 medium, 80% seeded confluence, and a 20X magnification). (C) This panel shows microscopy artifacts of water droplets on the interior of a sealed plastic bag containing a 24-well anoxic plate. <a href="https://www.jove.com/files/ftp_upload/58049/58049fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58049/58049fig3.jpg" /> 
Figure 3: Ratio of anoxic to normoxic HeLa 229 viable cells post-17 days incubation. This panel shows the effect of the seeding density on the HeLa 229 cell viability in the PS-74656 medium as compared to the normoxic control. The results are shown as the mean &#177; SEM.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58049/58049fig4.jpg" /> 
Figure 4: Ratio of anoxic to normoxic Vero viable cells 17 days post-incubation. This panel shows the effect of the seeding density on the Vero cell viability in the PS-74656 medium as compared to the normoxic control. The results are shown as the mean &#177; SEM.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Biology

10.0K Views.  Midwestern University. 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. 

Video: Anaerobic Growth and Maintenance of Mammalian Cell Lines

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.

Barwy Pasażowanie ludzkich embrionalnych komórek macierzystych linii Trypsyna

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

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Długoterminowe Imaging komórkach ssaków przy dużym polu Mikroskopia

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 ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...