Name: monitoring neuronal survival via longitudinal fluorescence microscopy
Uploaded: 2019-01-19T15:00:00
Description: Watch this Scientific Journal Video about Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy at JoVE.com

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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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 ...

This protocol enables researchers to monitor survival on a single-cell basis and identify variables that significantly predict cell death or survival. Conventional cytotoxicity assays assess populations of cells and lack the ability to discriminate individual cells. Longitudinal microscopy allows the assignment of time of death for each cell in a population.This technique is well-suited for evaluating new therapies for neurodegenerative diseases and other conditions. This method has proven invaluable for exploring disease mechanisms in neurodegenerative conditions, and could be applied with equal efficacy towards other disorders in which cell death is of interest. Researchers may struggle with achieving adequate transfection efficiencies without undue toxicity.Practice with the multichannel pipet and familiarity with the protocol should diminish these difficulty over time. Microscopy is an inherently visual methodology. Therefore, the technique may be more easily understood by watching in addition to reading.To begin, combine the appropriate amount of reduced serum media and transfection reagent in one tube, and reduced serum media and DNA in a separate tube. Incubate them at room temperature for five minutes. Then, combine the contents of both tubes and incubate at room temperature for 20 minutes.Use a multichannel pipet and steril plastic troughs to wash the cortical neurons two times with 100 microliters of neural basal media per well. And store the conditioned media and other remaining medias at 37 degrees Celsius. Replace the NBM with 100 microliters of previously prepared NBKY solution per well.After 20 minutes, pipet 50 microliters of the transfection reagent and DNA mixture dropwise into each well. Incubate the cells at 37 degrees Celsius for 20 minutes. Rinse the cells two times with NBKY solution.Then add 100 microliters of conditioned media and 100 microliters of NBC solution to the wells. To start imaging the cells, place the 96 well plate on a fluorescent microscope. Use a fiduciary mark unique to each plate as a reference for future alignment, and save an image for future reference.Then, navigate to areas of interest and record their x and y coordinates relative to the mark. Use a fiduciary mark unique to each plate as a reference for future alignment, and save an image for future reference. Finally, focus on the transfected cells expressing a fluorescent label.Take multiple images at regularly spaced intervals in the appropriate fluorescent channels. To begin with image processing, double click on the Fiji icon. Then, click and drag the image underscore processing macro onto the Fiji bar to open the macro within Fiji.Adjust lines two to seven of the image underscore processing macro according to the text protocol. Then, click on stitching, and then grid/collection stitching. Adjust the settings within the dropdown menus, type, and order, until an accurately stitched image is produced.Adjust grid type and stitch order variables in lines eight and nine according to these selections. Then, adjust line 10 to specify the number of images per well and set the background subtraction option in line 14 to true, if necessary. Adjust line 15 to set the rolling ball radius according to the text protocol, and click Run.Finally, open the output image stacks and manually identify the dead neurons according to the text protocol. Record the data in a spreadsheet file. To perform statistical analysis, double click on the icon for the survival.R script. Highlight line two and click the run button to load the survival library. Then, change the path in line five to call the input spreadsheet, and click the run button.Highlight lines eight and nine, and click the run button to perform the Cox proportional hazards analysis. Highlight lines 12 to 16 and 19 to 24. Click the run button to plot the cumulative risk of death and the survival data as a Kaplan-Meier curve.In this study, rat cortical neurons were transfected at four days post plating with a plasmid encoding the fluorescent protein M apple. 24 hours post transfection, neurons were imaged by fluorescence microscopy every 24 hours for 10 consecutive days. Following image acquisition, images were processed and were examined sequentially to mark the cell death.Time of death for each cell was determined by changes in fluorescence, morphology, and fragmentation of the cell body. Cox proportional hazards analysis performed on the survival data of the mutant neurons resulted in a hazard ratio of 2.2. This result indicated a faster rate of death in the mutant neurons in comparison to the wild type population.The most important thing to remember when attempting this procedure is to pipet carefully. Minimizing air exposure and avoiding bubbles is critical to a successful transfection. Following transfection, longitudinal fluorescence microscopy can be used to track various factors that may contribute to cell death, including protein localization, turnover, and expression level, in addition to cell survival.Within the field of neuro degeneration, the dynamic nature of longitudinal microscopy has shed light on whether the aggregation of disease-associated proteins represents a toxic or protective event. None of these reagents or instruments are hazardous.

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