Astrocytes have an important role in controlling oxidative stress of the central nervous system, helping maintain a homeostatic environment for neurons, as well as protecting neurons from reactive oxygen species. Here a method for assaying oxidative stress in astrocytes is presented. To illustrate how this assay works, hydrogen peroxide and ROS typically produced during cellular metabolism is applied to feline astrocytes next to assay.
For reactive oxygen species, the cell permeable indicator CMH 2D CFDA is added to the cells. CMH 2D CFDA enters the cell as a non fluorescent compound there cellular esterases remove the acetate groups. If reactive oxygen species are present, the compound is oxidized and becomes fluorescent.
The cells are then harvested and fluorescence is assay by flow cytometry. The number of fluorescent cells indicates the number of cells that are in an oxidative state. Aim, of course, is then to find to detect which specific oxygen radicals are present in the cells by using different access.
This is particularly very important in neurology and basically paved the way for new avenues of research neurologists because we can basically relate different diseases and different agents with different radicals produced in a neurological disease. Part of this job is due to the technique master by Omar Alvarado, Andrea Warnell, which are our two lab technicians. Wonderful, and our great Jo Jonathan Lee, which is a graduate student.
Begin this procedure by culturing the feline astrocyte cell line G 3 55 dash five in a 75 centimeter squared flask with DMEM plus nutrients. Incubate the flask at 37 degrees Celsius and 5%carbon dioxide until the cells are about 60%confluent, which should take approximately five days. Replace the medium every one to two days or as needed to maintain a healthy culture.
Once the cells reach the appropriate density. Remove the medium by aspiration. Add two milliliters of 0.25%Tryin lift the cells by pipetting up and down for 45 seconds to one minute at room temperature.
This helps minimize cell death and damage. Quickly transfer the suspension to a 15 milliliter conical tube containing 10 milliliters of medium mixed gently by flicking the bottom of the tube. Centrifuge the cell suspension at 300 GS 23 degrees Celsius for three minutes to obtain a pellet.
Following the spin. Aspirate the supernatant with a transfer pipette without disturbing the pellet. Then to the pellet at six to seven milliliters of fresh medium, and flick the tube to resuspend the cells.
Add one milliliter of cells to each well of a six well plate. Then add two to three milliliters of medium to each. Well place the plate in a 37 degree Celsius 5%carbon dioxide incubator.
Incubate the cells until they are 80 to 90%confluent roughly two to three days on the day of the experiment. Prepare a 100 micromolar solution of hydrogen peroxide in medium. Label the plate according to the samples that will be prepared.
The first well will contain a negative control, so only DMEM will be added and the cells will be double stained with CMH 2D CFDA and propidium iodide. The second well will function as a positive control DMEM with peroxide will be added and the cells will be double stained. The treatment condition in DPBS will be in the third well and will be double stained.
The fourth through sixth wells will contain flow controls. All of these cells will be untreated. Those in, well four will be unstained.
The cells in well five will be stained with CMH 2D CFDA only while the cells in well six will be stained with propidium iodide only. Remove the medium from each well of the plate. Then to wells 1, 4, 5, and six.
Add two milliliters of fresh medium to well. Two at two milliliters of the prepared hydrogen peroxide solution for the positive control. Then two L three.
Add two milliliters of DPBS for the treatment group. Incubate at 37 degrees Celsius, 5%carbon dioxide for three hours. Following the incubation, remove the solutions from the first.
Well add one milliliter of trypsin and pipette up and down to detach the cells. Transfer the cells to a two milliliter micro centrifuge tube containing one milliliter of DPBS. Then repeat the trypsin ization for the next.
Well, this step should be performed on each well individually in order to prevent cell death from prolonged exposure to trypsin. After all of the cells have been transferred to micro centrifuge tubes, spin them at 300 Gs for three minutes following the spin. Discard the supernatant and resuspend the cells in one milliliter of DPBS.
Next, CMH 2D CFDA is used to stain for reactive oxygen species. All steps for staining should be carried out with minimal light exposure to prevent bleaching. Begin by adding five microliters of a 10 millimolar CMH 2D CFDA solution to the appropriate samples.
Wrap the tubes in aluminum foil. Incubate them at 37 degrees Celsius, 5%carbon dioxide for 15 to 30 minutes. During the incubation, prepare the flow cytometer here.
The samples will be analyzed on a Beckman Coulter FC 500, equipped with a 488 nanometer argon laser. And the following band passes 5 25, 7 75, and six 20 nanometers, all plus or minus 20 nanometers. Before turning on the equipment, check the level of sheath and cleanse fluids.
Start the cytometer software, turn on the equipment and allow it to warm up for at least 20 minutes. During this time, check the pressure to ensure the equipment is running to the manufacturer's standards. Once the cells have been stained, spin them at 300 Gs for three minutes.
To obtain a pellet, aspirate the supernatant. Wash the samples with two milliliters of DPBS to remove any residual stain after spinning, again, aspirate and re suspend the cells in 0.5 milliliters of DPBS. Add one microliter of propidium iodide to the appropriate samples.
Next, transfer all of the samples to five milliliter flow cytometer tubes. The samples are now ready for analysis To clean the equipment, run one tube with 10%bleach and three tubes with nano pure water in positions one through four of the carousel. Select the cleanse protocol and run.
The cleanse solutions can be saved and reused. For the final cleanse for the calibration mix, flow check and PC seven solutions. Then to a tube containing 0.5 milliliters of water at 10 drops of the flow check solution and three drops of the PC seven solution.
Place the tube in position one of the carousel. Go to the tools menu and select auto set up scheduler. Select QC one L flow check C and run.
In the software. Generate a protocol that has the following histo plots, forward scatter or fs, which indicates cell size versus side scatter or ss, which indicates cell density PI or FL two versus CMH two, D-C-F-D-A, or FL one. Also set up the following histograms, cell number versus FL one, FL two, and FL three, which corresponds to the stains from the menu.
In the software. Select the protocol to be used. Then input the number of samples to the protocol.
Then under sample ID two, number each of the samples, place the samples in the carousel and run the protocol. The protocol shown here captures 10, 000 events for up to 180 seconds before switching to the next sample when finished, collecting data for all of the samples, clean the equipment as before, oxidative stress was assessed in healthy feline astrocytes using the method described in this video and the results were analyzed using Flo Jo 7.6. As shown here, an increase in reactive oxygen species was observed when hydrogen peroxide was added to the cells as indicated by the increase in fluorescence intensity, which is seen as a shift from the left to the right of the histogram.
Data from FL one was used to measure the intensity of CMH 2D CFDA, which is increased with oxidation indicating the presence of reactive oxygen species. As shown here. When healthy cells were treated with DMEM versus DPBS, there was no significant change in the amount of reactive oxygen species.
Another advantage we using flow cytometry as a technique is that it can be mastered in 45 minutes or one hour. Another technique that we are using and we're going to use is a fluorescence microscopy, which gives very good information about the components inside the cells. Like where are these oxid radicals located inside the cells?
There are special stains for it, and it can be very informative effect of the oxygen radicals on the neurological system. And this is important because sometimes this effect are associated with the use of drug abuse like methamphetamines.
