The overall goal of this procedure is to provide sensitive techniques for monitoring intracellular ROS levels and senescence-associated secretory phenotype, or SASP, during cellular senescence. Here, we demonstrate that these methods successfully reveal the increases in the levels of intracellular ROS and the SASP factors IL-6 and IL-8 during H-Ras-induced senescence in WI-38 normal human fibroblasts. These techniques allow monitoring changes in intracellular ROS levels during cellular senescence and the analysis of the expression of SASP factors.
The main advantage of this method is that they can be applied to many different cellular senescence models. Therefore, this method can ultimately help us to understand the role of ROS and SASP in cellular senescence and to study the underlying regulatory mechanisms. Demonstrating the procedure will be Young Yeon Kim and Jee-Hyun Um from my laboratory.
Begin this procedure by preparing the H-Ras retrovirus. First, seed ecotropic BOSC cells in a poly-L-lysine-coated culture dish, and culture at 37 degrees Celsius in a tissue culture incubator for one day. On the next day, transfect the BOSC cells with H-Ras retroviral DNA and packaging DNAs using 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 WI-38 cells in a 60-millimeter culture dish one day before harvest of H-Ras retrovirus, and culture for one day at 37 degrees Celsius in an incubator. On the next day, remove the growth medium, and add one milliliter of H-Ras virus media. Add an additional one milliliter of growth medium containing two microliters of polybrene stock solution, and incubate for one day at CO2 incubator.
At 24 hours after infection, remove the virus media, wash the cells twice with PBS, and add the growth media. To remove non-infected cells, treat the cells for two days with two micrograms per milliliter of puromycin. Prepare fresh SA beta-gal staining solution by diluting the stock solution.
Remove the culture media, and wash the cells once with PBS. Add fixation solution containing formaldehyde, and incubate for five minutes. Remove the fixation solution from the cells, and wash with PBS.
Add freshly prepared SA beta-gal staining solution to cover the dish. Seal the dish with Parafilm to avoid drying the sample, and incubate at 37 degrees Celsius in an incubator without CO2. Check the staining status of the cells under microscope on the next day.
SA beta-gal-positive cells will appear blue in the perinuclear region. Acquire an image of SA beta-gal staining with a CCD camera, and calculate the percentage of SA beta-gal-stained cells by counting the number of SA beta-gal-positive cells among at least 200 total cells. Plate two times 10 to the fifth WI-38 cells on a 60-millimeter culture dish, and induce senescence by infecting the cells with H-Ras retrovirus as described previously.
At the desired time point for measurement of ROS, remove the growth medium and wash once with PBS. Detach the cells by treatment with trypsin/EDTA solution, and determine the cell count. Transfer one times 10 to the fifth cells into a 15-milliliter conical tube, and collect the cells by centrifugation.
Remove the growth medium carefully, and add one milliliter of freshly prepared DCF-DA staining medium. Carefully resuspend the cell pellet, and incubate at 37 degrees Celsius for 30 minutes in the dark. Collect the cells by centrifugation and wash once with PBS.
Resuspend the cells with one milliliter of PBS, and transfer the samples into a FACS tube. The DCF-DA fluorescence signal can be measured using a flow cytometer equipped with a 488-nanometer blue laser. First, run a non-stained control cell sample.
In the dot plot of the forward scatter versus side scatter, select live cells and eliminate dead cells and cell debris by using a gating tool. Next, in the histogram displaying DCF-DA fluorescence signal, adjust the blue laser voltage to place the peak from the control cells at the left edge. Now, run each sample stained with DCF-DA, and record at least 10, 000 events.
Induce senescence by infecting the cells with H-Ras retrovirus as described previously. At one day before harvest, wash the cells twice with PBS. Add three milliliters of DMEM without FBS, and culture the cells at 37 degrees Celsius in a tissue culture incubator for one day.
After 24 hours, collect the conditioned medium, and store at negative 80 degrees Celsius for subsequent ELISA. Trypsinize the cells, and harvest the cells in a 1.5-milliliter microcentrifuge tube by centrifugation. Prepare total RNA using the conventional RNA extraction method, and generate cDNA from two micrograms of total RNA through a reverse transcription reaction.
For real-time PCR, prepare SYBR Green PCR Master Mix for all reactions containing the forward and reverse primers for IL-6 or IL-8. Prepare the reaction mixture for each sample containing actin primers as an internal control. Add 48 microliters of Master Mix and two microliters of cDNA products diluted one to three to each well in a 96-well optical qPCR plate.
Perform each reaction in triplicates. Set up the real-time PCR reaction program on the thermal cycler, and perform real-time PCR. Upon completion of the run, collect the threshold cycle values, or CT values, and calculate the mRNA levels of IL-6 or IL-8 after normalization to the actin levels.
Thaw conditioned media harvested previously from senescent WI-38 cells. Remove cell debris by centrifugation at 500 g for five minutes. Concentrate the conditioned medium by 10-fold using a centrifugal filter with a pore size of 3, 000 dalton.
Perform the ELISA with the appropriate IL-6 or IL-8 ELISA kit. First, coat the ELISA plate well with diluted IL-6 or IL-8 antibody by incubating the plate overnight at room temperature. After washing each well four times, add blocking buffer to each well, and incubate for one hour.
Wash the plate four times. Add either serially diluted ELISA standards or concentrated conditioned medium samples, and incubate for two hours. After washing the plate, incubate the samples with a secondary antibody and streptavidin-HRP conjugate sequentially.
Add 100 microliters of TMB substrate solution to each well, and incubate at room temperature for 20 minutes to allow for color development. Stop the reaction by adding stop solution. Read the absorbance of each well with an ELISA reader at 450 nanometer.
Plot the standard curve for the generated standards. Calculate the concentrations of IL-6 and IL-8 in the conditioned medium according to the standard curves. Plot the results after normalization to the cell count.
Infection of WI-38 normal human fibroblasts with H-Ras retrovirus induced dramatic changes in cellular morphology. SA beta-gal staining activity was observed in more than 70%of the cells, indicating that H-Ras expression induces cellular senescence in WI-38 cells within six days. DCF-DA staining analysis reveals that H-Ras expression also induces a significant increase in intracellular ROS levels.
The increase in intracellular ROS levels is observed as early as two days after H-Ras expression and is maintained until the six-day time point. Real-time PCR analysis of the representative senescence-associated secretory factors IL-6 and IL-8 reveals that mRNA expression of IL-6 and IL-8 is remarkably increased in senescent WI-38 cells. ELISAs of conditioned medium confirmed that IL-6 and IL-8 secretion is also increased in H-Ras-induced senescent cells.
Inducing cellular senescence through oncogene expression, DNA damage, or oxidative stress usually takes six to eight days. Once the cells are ready, ROS measurement can be conducted within two hour if performed properly. After mastery of these techniques, analysis of the mRNA and protein levels of SASP factors can be completed within one day.
When analyzing ROS levels and SASP induction, it is important to confirm cellular senescence through SA beta-gal staining and additional method if necessary. In addition, it is critical to remember that the increase of ROS occurs in the early phase of senescence, whereas the development of SASP is detectable only after senescence is fully established. These techniques can be applied to a wide variety of cellular senescence models.
After watching this video, you should have a good understanding of how to analyze ROS levels and SASP induction during cellular senescence.
