This method can help to measure the effect of hypoxia/reoxygenation on cells in the mitochondrial membrane potential, and it can be used to evaluate if an agent is protective against a injury. The main advantage of this assay is that MMP detected by it is in the event of factors such as mitochondrial size. Additionally, it has a low requirement for materials and the radiance.
Begin with 70%confluent human cardiac myocytes in a six well plate. After washing the cells with phosphate buffer saline, add drug or vehicle in two mililiters of serum and glucose free DMEM. Incubate in a cell culture incubator containing 5%CO2 and 95%air at 37 degrees Celsius for 30 minutes prior to hypoxia.
After 30 minutes place the dish of human cardiac myocytes into a 2.5 liter airtight anhypoxic jar. Fitted with a catalyst to scavenge free oxygen, and an anerobic indicator to measure the oxygen tension in the medium. Place the jar in the tissue culture incubator.
The color of the anerobic indicator changes from light blue in normoxic environment to pale white in hypoxic conditions. Reoxygenate the human cardiac myocytes by removing the six well plate from the jar and placing it on the incubator shelf for two hours. Following the reoxygenation, rinse the cells with one milliliter of pre-warmed PBS, and add 0.5 mililiters of pre-warmed 25%trypsin along the wall of the well.
Incubate at 37 degrees Celsius for about one minute until all human cardiac myocytes are almost detached. Then add one milliliter of DMEM complete medium to the wells to neutralize the trypsin. Transfer the cell suspension to a 15 milliliter centrifuge tube, and pellet HCMs by centrifuging at 500 times G for three minutes, at room temperature.
Following centrifugalization, carefully aspirate the supernatant without disturbing the pellets. Then add 5 milliliters of human cardiac myocyte complete medium, and 5 milliliters of JC-1 working solution to each tube. Resuspend the HCMs by pipetting up and down.
Cover the tubes with aluminum foil to prevent bleaching of the fluorescent indicator, and incubate the cells in normal conditions for 20 minutes. After pelleting the human cardiac myocytes by centrifuging at 500 times G for three minutes in a centrifuge pre-cooled to four degrees Celsius, rinse cells twice with two milliliters of ice cold JC-1 staining buffer. After the second rinse, resuspend the human cardiac myocytes in 300 microliters of ice cold staining buffer.
And filter the samples into the respective sample tubes. Analyze the cells by flow cytometry within 30 minutes. Start the flow cytometer and ensure that the software is connected.
Perform fluidic start up. Start the stream, and set up the break off. Open two dot plot windows in the flow cytometry software.
Select forward scattered light on the X-axis, and side scattered light on the Y-axis in the first dot plot. To represent the characteristics of the cells in terms of their size an their granularity, respectively. Select the PE detection channel for the Y-axis.
And FITC for the X-axis, using a logarithmic scale in the second dot plot, which is used to measure the florescence intensity of JC-1 dye in the cells. Click unload to let out the tube support arm, and position the blank sample tube containing human cardiac myocytes that have not been dyed with JC-1. Click load to return the support arm to its working position.
Click record to collect particles from suspension in the blank sample tube. And adjust voltages of flouresence channels in the second dot plot. Gate the cell population for further analysis in the first dot plot.
Next, analyze the human cardiac myocytes. Display the statistics of each sample tube and calculate the MMPs of each sample by dividing the ratio of red flouresence intensity by that of green flouresence intensity. Hypoxia and reoxygenation significantly induce apoptosis of human cardiac myocytes, compared to normal cells as measured by the Annexin PI ratio.
a Chinese traditional medicine with cardio-protective effects, prevented the apoptosis of HCMs after hypoxia/reoxygenation. As shown in here, the JC-1 red/green ratio after treatment with the mitochondrial CCP was almost zero. Consistent with the results from the apoptosis assay.
The H/R treated group displayed a significantly lower red/green ratio, compared with the normal group. And reversed such a ratio change. While attempting this procedure, it's important to remember to start the cells loaded with JC-1 in ice cold standing buffer, as temperature has a considerable effect on the stabilities of mitochondrial membrane potential.
Following this procedure, other methods like genetic manipulation can be performed to answer additional questions like if the decrease of MMP results from the opening of MPTP.
