Name: viability assays for cells in culture
Uploaded: 2014-01-20T13:45:00
Description: Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

We acknowledge Juliann Jaumotte for the idea of saving on the volumes of reagents in the ATP assay. We are deeply grateful for the superb administrative support of Mary Caruso, Deb Willson, and Jackie Farrer and to the Mylan School of Pharmacy for providing financial support for these studies. Thanks are also due to the Hunkele Dreaded Diseases Foundation and the Parkinson&#8217;s and Movement Disorders Foundation for their financial support of the primary neuronal studies.

A schematic of the protocols is illustrated in Figure 1.
1. Cell Plating
Plate cells in 96-well plates at different plating densities (Figure 2). For linearity checks on the N2a neuroblastoma cell line, plate 2.5k, 5k, 10k, and 15k cells per well in 3 or 6 wells/group. For linearity checks in rat primary cortical neurons, plate 25k, 50k, 100k, and 200k cells per well in 3 or 6 wells/group. If the cell lines or primary cells of interes...

We have found that signal strength in all three viability assays is linear and correlated with plating density. However, not all the assays are equally sensitive to 2-fold or 1.5-fold changes in plating density. For N2a cells, the infrared assays are less sensitive than the ATP assay, particularly at lower plating densities. Although the infrared assays are less sensitive than ATP, the DRAQ5 + Sapphire assays and the &#945;-tubulin assays are in good agreement in that they reveal the highly protective impact of N-acetyl ...

The most common viability assay in the biological sciences involves cell counts. This is evidenced by an analysis of the top (most recent) 200 publications that appeared in PubMed with either of the keywords &#8220;in vitro&#8221; or &#8220;culture&#8221; on 4/29/2013 and 4/30/2013. Of these publications, 23.5% used cell count assays, including manual cell number counts, automated cell number counts with imaging software, and Trypan blue exclusion. The Live/Dead assay was used in 1% of these publications. The number of publications using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for metabolic viability was 11%. This survey of the literature also shows that the number of publications using assays such as MTT in conjunction with cell count assays was only 3.5%. Despite the trend to use one viability assay by itself, assessing cellular function in combination with cell number seems the best choice for assessing cellular integrity. Cell counts by themselves are not sufficient because the remaining cells may not be functional or healthy even though they are present in the well1,2. Conversely, functional measures such as ATP may increase or decrease in the absence of parallel changes in the number of cells. The uncoupling of metabolic readouts from cell number suggests that ATP and MTT assays should never be used as the sole viability assay. In the present report, three viability assays that survey both cellular structures and metabolic function are described, for a more comprehensive view of cellular integrity than any one assay by itself can afford.

Two of our assays require an infrared imager that measures fluorescence in the 700 and 800 nm channels. Noise is low in the infrared wavelengths, leading to higher signal-to-noise ratios3. The Odyssey imager that we use has a 4.5 log dynamic range and a bit-depth of 16, translating to 216 or 65,536 shades of infrared. This can be contrasted to 8-bit color imaging, which only affords 28 or 256 shades of color for each wavelength. Thus, 16-bit imaging has finer resolution. It should be noted that the original infrared images are often pseudocolored green (800 nm) and red (700 nm) in published reports for presentation. Odyssey imagers are commonly used both for Western blotting and In-Cell Westerns4-7. In-Cell Westerns on formaldehyde-fixed cells use primary antibodies against any protein of interest and label them in turn with infrared fluorescent secondary antibodies. This technique is known to be particularly useful for phosphorylation endpoints6. In our In-Cell Westerns, we stain fixed cells for cytoskeletal proteins &#945;-tubulin or the neuronal microtubule associated protein 2 (MAP2) in the 800 nm channel. These proteins are abundant enough to yield high signal-to-noise ratios. We also stain our plates in the 700 nm channel for nuclei with the DRAQ5 stain and for the cytoplasm with the Sapphire stain. Both the cytoskeletal proteins and the DRAQ5 + Sapphire stains thus reflect cellular structures.

The third viability assay measures metabolic function and is called &#8220;Cell Titer Glo.&#8221; In this luciferase-based assay, luminescence values are in direct proportion to ATP levels. ATP assays are commonly used to quantify viable cells8-12. However, including the word &#8220;titer&#8221; in the name of the assay is somewhat of a misnomer because ATP output per cell can change as a function of toxin treatments and is therefore not always in proportion to cell number8. ATP levels can also be affected by circadian rhythms13 and by cell division14 and cell differentiation15. Nevertheless, the ATP assay shown here is simple to perform and useful because ATP is a robust measure of metabolic viability16-21, if not cell number per se. Using this assay to complement the infrared In-Cell Westerns therefore yields a more comprehensive picture of cellular integrity than any one assay alone.

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		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Cell Titer Glo</td>
			<td style="width:112px;">Promega</td>
			<td style="width:93px;">G7572</td>
			<td style="width:507px;">Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 &#176;C for at least 21 weeks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">18% Formalin</td>
			<td style="width:112px;">Thermo-Shandon</td>
			<td style="width:93px;">9990244</td>
			<td style="width:507px;">Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sucrose</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:507px;">It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Odyssey Block</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">927-40003</td>
			<td style="width:507px;">This fish serum can be bought in bulk and frozen at -20 &#176;C for long term use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Triton-X 100</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">21568</td>
			<td style="width:507px;">We store a stock solution of 10% Triton-X 100 in sterile water at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Monobasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S468</td>
			<td style="width:507px;">One can also buy PBS tablets or 10x PBS solutions, but they are more expensive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Dibasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S373</td>
			<td style="width:507px;">See above</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Azide (250x)</td>
			<td style="width:112px;">Ricca Chemical Company</td>
			<td style="width:93px;">7144.8-16</td>
			<td style="width:507px;">Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 &#176;C until they become contaminated with debris</td>
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		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-&#945;-tubulin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">T5168</td>
			<td style="width:507px;">This antibody is expensive but can be greatly diluted and is highly specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-MAP2</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">M9942</td>
			<td style="width:507px;">This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">800 nm Goat anti-mouse IgG</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">926-32210</td>
			<td style="width:507px;">Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">DRAQ5</td>
			<td style="width:112px;">Biostatus</td>
			<td style="width:93px;">DR50200</td>
			<td style="width:507px;">This compound used to be sold by LI-COR at 1 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sapphire</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">928-40022</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Luminometer</td>
			<td style="width:129px;">PerkinElmer</td>
			<td style="width:189px;">VICTOR3 1420 multilabel counter</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Odyssey Imager</td>
			<td style="width:129px;">LI-COR</td>
			<td style="width:189px;">9201-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Shaker/Mixer</td>
			<td style="width:129px;">Research Products International</td>
			<td style="width:189px;">248555</td>
			<td></td>
		</tr>
	</tbody>
</table>

viability assays for cells in culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

The rate-limiting factor in these experiments is the infrared staining, as the ATP assay is relatively brief in duration. For the infrared assays, we anticipate that eight 96-well plates can be stained and scanned within one day by staggering two batches of four plates each (see Figure 1). This estimation assumes 20 min of fixation, 30 min of washing, 30 min of blocking, 2 hr primary antibody incubation followed by 30 min of washes, 1 hr secondary antibody incubation followed by 30 min of washes, 30 min ...

Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

Viability Assays for Cells in Culture

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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