Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.
The overall goal of these techniques is to provide a toolbox for enabling the induction, monitoring, and quantification of cellular senescence. These methods can help answer key questions about the identification and quantification of cellular senescence and the biologic factors that regulate cellular and organismal aging. The main advantage of these techniques is that they encompass a variety of methods for monitoring cellular senescence in different models, tissues, and cell types.
Begin by plating the early passage proliferative cells of interest at a sparse density in the appropriate dish for the analysis. And culture the cells overnight in a tissue culture incubator. When the cells are 50-60%confluent, aspirate the growth medium and wash the cells with PBS two times.
After the second wash, cover the cells with a thin layer of PBS. And expose the cells to a single 10 grade dose of gamma irradiation. Replace the PBS with growth medium.
And return the cells to the incubator. Changing the medium every 48 to 72 hours with daily monitoring for morphological changes under a light microscope. Seven to 10 days after the irradiation, thaw all of the reagents from a commercial senescence associated beta-galactosidase assay kit in a 37 degree Celsius water bath.
When all of the reagents reach room temperature, prepare the fixative and staining solutions according to the manufacturer's instructions. And gently wash each well with room temperature PBS. Take care to rinse the cells carefully as senescence cells do not adhere well and can be easily removed during washes.
Fix the cells in each well with one milliliter of fixative solution per well for 10 to 15 minutes at room temperature. Followed by two two milliliter washes with PBS per well. Next, add one milliliter of beta-galactosidase staining solution to each well and cover the plate with aluminum foil for a 24 to 48 hour culture in a 37 degrees Celsius incubator without carbon dioxide.
Check the cells at 24 hours. When the cells are sufficiently stained, replace the beta-gal solution with fresh PBS and examine the cultures on a light microscope with an attached camera at a 10x or a higher objective. The senescence associated beta-galactosidase cells will appear blue.
Then, obtain at least five non-overlapping images per well. And manually count the total number of senescence associated beta-galactosidase positive cells and the total number of cells per image to calculate the percentage of senescence associated beta-galactosidase cells per well. 10 days after gamma irradiation, wash the cells three times with PBS.
After the last wash, replace the PBS with 2.5 milliliters of serum free medium, supplemented with antibiotics per six centimeter dish and return the cells to the tissue culture incubator overnight. The next morning, transfer the medium into a conical tube on ice. And wash the cells two times with fresh PBS.
Then, trypsinise and count the cells. Next, centrifuge the supernatant harvested from the cell culture plate. And then filter it through a 0.45 micrometer syringe strainer and freeze the medium in 500 microliter aliquots at 80 degree Celsius until their analysis by ELISA.
Increased senescence associated beta-gal staining occurs with replicative and DNA damage induced senescence. Note the enlarged flat shape of the senescent cells compared to the spindle-like appearance of the proliferating fibroblasts. In this experiment, proliferating young or replicative senescent cells were fixed as described in the written protocol.
And stained with an anti-body against a bio marker for DNA double strand breaks and DAPI. With an apparent increase in the number of DNA double strand break fosi observed in the senescent cells. RT-qPCR analysis of senescence associated secretory phenotype factors reveals that at least some factors are up regulated after gamma irradiation induced senescence in low passage human diploid fibroblasts at the MRNA level.
Which is further confirmed by ELISA of the gamma irradiated cell culture supernatants. Once mastered, the DNA damage induction and SA beta-gal staining can be completed in approximately 30 minutes of hands-on time. The condition media for quantifying SASP protein levels can be prepared in about 30 minutes.
While attempting this procedure, it's important to remember to monitor them undergoing morphological changes in the cell cultures before proceeding to the senescent marker quantification. After its development, these techniques paved the way for researchers in the fields of aging and cancer to explore senescence in a variety of tissues, cell cultures, and model organisms. After watching this video, you should have a good understanding of how to induce, monitor, and quantify cellular senescence.
