Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

This procedure allows oligo DDR glial cell death to be monitored in real time. This is accomplished by first culturing the cells, then the cells are prepared for live cell imaging. The next step is to provide a treatment to the cells and monitor the effects using fluorescence microscopy.

The final step is to perform data analysis. Ultimately, results show rates of cell death in a cell population following a treatment or stimulus by monitoring nuclear fluorescence from a caspase activated fluro dye. The main advantage of this technique over existing methods like immuno staining, is the ability to gather multiple time points simultaneously.

This method can help answer key questions in cell biology, such as responses to pharmacologic, reagent, and various treatments. Now, this method can provide insight into death within cell cultures. It can also be applied to other systems such as ex vivo, organotypic tissues, including brain slices.

Generally, individuals new to this method will struggle as they adjust to working with live tissue rather than fixed tissue. Visual demonstration of this technique is important as there are a lot of steps to prepare live cell samples compared to fixed tissue. First thaw frozen immortalized N 19 oligo droog glial cells in a 37 degree Celsius water bath while the cells are thawing.

Add seven milliliters of high glucose DMEM supplemented with 10%FBS and 1%penicillin streptomycin to a 10 centimeter culture dish. Then add the cell suspension dropwise to the plate and gently agitate the Petri dish to disperse the cells evenly. Culture the cells at 34 degrees Celsius in a 5 cent carbon dioxide incubator for four hours after four hours.

Aspirate the media from the plates to remove any remaining DMSO.Cryoprotectant. Replace with seven milliliters of fresh media after four to seven days of growth. When the cells have reached 70 to 80%Confluence aspirate the media and pipette one milliliter of drips in onto the cell sheet, incubated room temperature for five minutes.

Then harvest the cells next seed, a 10 centimeter tissue culture dish with the harvested cells at a density of 0.1 times 10 to the six cells per milliliter, and place this dish in the tissue culture incubator. After one further massage, the cells can be plated for live cell imaging experiments. To do this, harvest the cells as before, then remove 30 microliters of the cell suspension and count the cells using a hemo cytometer.

Next, use sterile forceps to place uncoated glass cover slips. In the wells of a six well plate add cells to the well at a density of 0.1 times 10 to the six cells per milliliter. In two milliliters of phenol free DMEM high glucose media supplemented with 10%FBS and 1%penicillin streptomycin allow the cells to grow overnight at 34 degrees Celsius and 5%carbon dioxide before transfection.

The next day, combine 100 microliters of serum free media 0.5 to four micrograms of purified plasmid, DNA and four microliters of FU gene hd. Gently vortex the mixture twice and then allow the DNA to complex at room temperature for five minutes. After the incubation time has elapsed, add FU gene HD DNA mixture directly to the cultured cells.

Tilt the plate gently to mix. Then culture the cells for an additional 48 hours at 34 degrees Celsius and 5%carbon dioxide prior to treatment or experimentation. First turn on the LCI Cham light live cell instrument control box.

This should be done three hours before the experiment begins to ensure thorough warming of the stage. At a minimum, the control box should be turned on one hour before beginning the experiment. Next, working in a flow hood, spray the LCI Cham light magnetic type culture chamber and forceps with 70%ethanol and allow them to dry for five minutes.

Remove the cells from the incubator and examine them under the microscope to confirm that they are healthy. The cells should appear a durant and well spread on the glass cover slip with numerous membrane processes. Cells with a reduced number of process extensions or and an irregularly shaped nucleus are likely stressed from transfection and are unsuitable for further experiments working in the flow hood.

Tilt the six well plate and remove the cover slip with forceps. Quickly place the cover slip cell side up into the bottom plate of the chamber. Do not allow the cover slip to dry.

Next, attach the magnetic main body of the culture chamber and add 500 microliters of media from the original six well plate onto the top of the cover slip. Reusing the media decreases the amount of stress placed on the cells caused by environmental changes and can also be useful for assessing extracellular secreted factors. After the media has been added, place the glass cover on the culture chamber and use a Kim wipes braid with 70%to remove any residual material that could interfere with microscopy from the bottom of the cover slip.

Place the culture chamber in the 34 degrees Celsius and 5%carbon dioxide incubator for 30 minutes. This step will ensure that the chamber itself warms the 34 degrees to reduce shifting of the cover slip. As the metal warms up, it is necessary to work with the ambient room lighting as low as possible during the following steps.

First, Thora previously prepared elequa of nuke view at room temperature. Then retrieve the culture chamber from the incubator and quickly place it in the environmental chamber of the microscope. Turn on the 5%pre-mixed carbon dioxide tank with dual regulator.

Next, using the 10 times objective with the microscope lamp set to around 2.5 volts and exposure time to 240 milliseconds. Focus using brightfield microscopy to visualize the cells on the computer monitor. Then add an 80 millimolar concentration of potassium ion or other apoptosis inducer to the cells via media exchange with slow and local perfusion using a peristaltic pump.

The nuke view 4 88 substrate is stable in cell culture for experiments lasting as long as 36 hours. Therefore, it is possible to conduct experiments over this duration of time. First set up the microscope to visualize fluorescence from red and green fluorescent protein.

Then click on the red channel to display the transfected cells. Select and save approximately 12 frames where there are several transfected cells and save these XY stage points. Have the microscope capture images in the brightfield red channel and green channel at these saved stage points every six minutes.

After collecting multiple XY points from duplicate or triplicate experiments, compile the data from separate experiments performed on different days and perform data analysis and statistical testing as described in the written protocol to compare the ratio of CASP bay's negative to CASP bay's positive cells. Nuke view 4 88 substrate can indicate an increased rate of apoptosis of N 19 oligo DDR glial cell cultures. Following a treatment with a high extracellular potassium concentration, these baseline images were acquired using a 10 times objective.

The N 19 cells were transfected with RFP and were either treated with three microliters nuke view, 4 88 substrate for control cells, or three microliters nuke view, 4 88 substrate and 80 millimolar potassium. These images illustrate that the number of apoptotic cells increases over time, three, six and nine hours after treatment with 80 millimolar potassium under control conditions, significant amounts of apoptosis were not observed compared to the 80 millimolar potassium treated cultures. The background green signal observed in the control conditions indicates the cells that are undergoing apoptosis without the addition of extracellular potassium.

A 12 hour time course is adequate for studies involving neurological insults After 12 hours, potassium treated cultures demonstrated approximately 45%cell death. Virtually none of the cells in the control experiment exhibit apoptosis by the 12 hour time point. Although in other situations, the experiments may be required to run longer.

This graph shows the increase in caspase three positive cells over the time course of the 12 hour experiment. Once pestered, this technique can be done in one hour and data can be collected overnight if done properly. While attempting this procedure, it's important to remember to be consistent between treatments Following this procedure.

Other methods such as immuno staining can be used to look at other downstream proteins involved in the pathways studied. After watching this video, you should have a good understanding of how to prepare samples for imaging. Acquire a data set and analyze your data.