We thank Steve Finkbeiner and members of the Finkbeiner lab for pioneering robotic microscopy. We also thank Dan Peisach for building the initial software required for image processing and automated survival analysis. This work was funded by the National Institute for Neurological Disorders and Stroke (NINDS) R01-NS097542, the University of Michigan Protein Folding Disease Initiative, and Ann Arbor Active Against ALS.

All vertebrate animal work was approved by the Committee on the Use and Care of Animals at the University of Michigan (protocol # PRO00007096). Experiments are carefully planned to minimize the number of animals sacrificed. Pregnant female wild-type (WT), non-transgenic Long Evans rats (Rattus norvegicus) are housed singly in chambers equipped with environmental enrichment, and cared for by the Unit for Laboratory Animal Medicine (ULAM) at the University of Michigan, in accordance with the NIH-supported Guide fo...

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The authors have nothing to disclose.

Here, methodology to directly monitor neuronal survival on a single-cell basis is presented. In contrast to traditional assays for cell death that are limited to discrete time points and entire populations of cells, this method allows for the continuous assessment of a variety of factors such as cellular morphology, protein expression, or localization, and can determine how each factor influences cellular survival in a prospective manner.
This methodology can be modified to fit a wide array of...

Abnormal cell death is a driving factor in many diseases, including cancer, neurodegeneration, and stroke1. Robust and sensitive assays for cell death are essential to the characterization of these disorders, as well as the development of therapeutic strategies for extending or reducing cellular survival. There are currently dozens of techniques for measuring cell death, either directly or through surrogate markers2. For example, cell death can be assessed visually with the help of vital dyes that selectively stain dead or living cells3, or by monitoring the appearance of specific phospholipids on the plasma membrane4,5,6. Measurements of intracellular components or cellular metabolites released into the media upon cellular dissolution can also be used as proxies for cell death7,8. Alternatively, cellular viability can be approximated by assessing metabolic activity9,10. Though these methods provide rapid means of assessing cell survival, they are not without caveats. Each technique observes the culture as a single population, rendering it impossible to distinguish between individual cells and their unique rates of survival. Furthermore, such population-based assays are unable to measure factors that may be important for cell death, including cellular morphology, protein expression, or localization. In many cases, these assays are limited to discrete time points, and do not allow for the continuous observation of cells over time.
&#160;In contrast, longitudinal fluorescence microscopy is a highly flexible methodology that directly and continuously monitors the risk of death on a single-cell basis11. In brief, longitudinal fluorescence microscopy enables thousands of individual cells to be tracked at regular intervals for extended periods of time, allowing precise determinations of cell death and the factors that enhance or suppress cell death. At its base, the method involves the transient transfection or transduction of cells with vectors encoding fluorescent proteins. A unique fiduciary is then established, and the position of each transfected cell in relation to this landmark allows the user to image and track individual cells over the course of hours, days, or weeks. When these images are viewed sequentially, cell death is marked by characteristic changes in fluorescence, morphology, and fragmentation of the cell body, enabling the assignment of a time of death for each cell. The calculated rate of death, determined by the hazard function, can then be quantitatively compared between conditions, or related to select cellular characteristics using univariate or multivariate Cox proportional hazards analysis12. Together, these approaches enable the accurate and objective discrimination of rates of cell death among cellular populations, and the identification of variables that significantly predict cell death and/or survival (Figure 1).
Although this method can be used to monitor survival in any post-mitotic cell type in a variety of plating formats, this protocol will describe conditions for transfecting and imaging rat cortical neurons cultured in a 96-well plate.

<table>
	<tbody>
		<tr>
			<td>Neurobasal Medium</td>
			<td>GIBCO</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>CompactPrep Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12863</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride Hexahydrate</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic Acid Hydrate</td>
			<td>TCI</td>
			<td>H0303</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly D-Lysine</td>
			<td>Millipore</td>
			<td>A-003-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>52887</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>GIBCO</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>TPP</td>
			<td>0876</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring neuronal survival via longitudinal fluorescence microscopy

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Using the transfection procedure described here, DIV4 rat cortical neurons were transfected with a plasmid encoding the fluorescent protein mApple. Beginning 24 h post-transfection, cells were imaged by fluorescence microscopy every 24 h for 10 consecutive days. The resultant images were organized as indicated in Figure 2, then stitched, stacked, and scored for cell death (Figure 1). Figure 3 shows a...

Watch this Scientific Journal Video about Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy at JoVE.com

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death.

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to ...

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6.0K Views.  University of Michigan School of Medicine. Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death.