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.
