The overall goal of this procedure is to provide a sensitive technique for measuring cellular mitophagy activity using flow cytometry. Here we demonstrate that this method was successfully used to assess the dramatic increase in HeLa cell mitophagy, after treatment with CCCP, a mitochondrial uncoupler. This technique allows mitophage activity to be measured very quickly and easily.
The main advantage of this technique is that you can sensitively measure mitophage activity in living cells without any additional staining. Therefore, this technique will ultimately be able to help in understanding the mechanism and physiological role of mitophage. Demonstrating the procedure will be Jee-Hyun Um and Young Yeon Kim from my laboratory.
Begin this procedure by preparing the mt-Keima, a mitochondria-targeting Keima lentivirus. First seed HEK293T cells in a poly-L-lysine coated culture dish and culture them at 37 degrees Celsius in a tissue culture incubator for one day. On the next day transfect HEK293T cells with mt-Keima lentiviral DNA and packaging DNase using a transfection reagent according to the manufacturer's instructions.
After eight hours of transfection remove the transfection media and add eight milliliters of fresh media. Collect the media containing viral particles 48 hours later and remove cellular debris by centrifugation. Next filter the virus-containing media with a 0.45 micrometer syringe filter.
Plate HeLa-Parkin cells in a 60 millimeter culture dish one day before harvesting the mt-Keima lentivirus and culture the cells with four milliliters of growth media for one day at 37 degrees Celsius in an incubator. On the next day remove the growth medium add one millimeter of mt-Keima virus media add an additional two milliliters of growth medium containing 3 microliters of polybrene stock solution and incubate the cells for one day in a CO2 incubator. At 24 hours after infection remove the virus media wash the cells twice with PBS and add four milliliters of growth media.
To remove uninfected cells treat cells for two days with two micrograms per microliter puromycin. Prepare fresh media containing 10 micromolar CCCP. Remove the cultured media and add four milliliters of fresh growth media containing CCCP.
Incubate the cells at 37 degrees Celsius in a CO2 incubator for six hours. Remove the growth medium and wash the cells once with PBS. Detach the cells by treatment with trypsin EDTA solution.
Collect the cells by centrifugation and re-suspend them in one milliliter of PBS. Transfer the cells in a FACS tube and place them on ice. For flow cytometry turn on the flow cytometer and start the analyzing software.
Generate a new experiment and select BV605 for the violet laser and PE-CF594 for the yellow-green laser in the parameter window. First run a control sample of cells not infected with the mt-Keima lentivirus. In the plot of forward scatter versus side scatter draw a gate, using the polygon icon, around the live cells and eliminate dead cells and cell debris.
Then run HeLa-Parkin cells infected with the mt-Keima lentivirus. In the dot plot of BV605 versus side scatter adjust the voltage so that HeLa-Parkin cells expressing mt-Keima are clearly distinguished from control cells that do not express mt-Keima. Next in the dot plot of PE-CF594 versus side scatter adjust the voltage so that HeLa-Parkin cells expressing mt-Keima are clearly distinguished from control cells.
Then in the dot plot of BV605 versus PE-CF594 select the mt-Keima-positive cell population by drawing a gate using the polygon icon. Next create another dot plot of BV605 versus PE-CF594 showing only mt-Keima gated cells. In this dot plot, draw a gate around untreated mt-Keima-positive cells.
This gate is referred to as the low gate. Then draw another gate containing the region above the low gate. This gate is referred to as the high gate because it contains cells with high mitophagy activity.
Then select the Show Population Hierarchy menu. The Percent of Parent value represents the percentage of cells in the high gate among the mt-Keima-positive population. Now run each sample and record at least 10 000 events in the mt-Keima stopping gate.
Flow cytometry analysis of untreated HeLa cells expressing mt-Keima showed a low mitophagy level. CCCP treatment induced a dramatic increase in mitophagy cells in the high gate. The percentage of mitophagy cells increased by more than 10 fold at six hours after CCCP treatment.
This increase in mitophagy by CCCP treatment was completely abolished by co-treatment with bafilomycin A.Once the cells expressing mito-Keima are ready mitophage measurement using flow cytometry can be conducted within one hour if prepared properly. For accurate mitophage analysis by flow cytometry it is important to set up the correct high and low gates. When analyzing new types of cells it is necessary to use well-known cells such as HeLa cells as a control.
In addition it is important to remember that mitophage should be fully confirmed by additional characteristics such as a decrease in mitochondrial protein and a reduction in mitochondrial DNA. This technique can be applied to a wide variety of cell types. After watching this video you should have a good understanding of how to analyze mitophage by flow cytometry.
