This method can help answer key questions in the aging and cancer field such as how to perform common techniques to induce cellular senescence and how to evaluate the senescence phenotype. The main advantages of this technique are that it is easy to follow, highly reproducible, does not require any special or expensive equipment, and it results in the induction of senescence in at least 50%of the cellular population. Demonstrating this procedure will be Alejandra Hernandez-Segura, a graduate student from our lab.
To begin, seed seven times 10 to the fifth viable primary fibroblasts with a low Population Doubling or PD in a T75 flask containing 10 milliliters of D10. Incubate the cells in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen overnight. Dilute 11 microliters of the 1, 000X doxorubicin stock solution in 11 milliliters of D10 to a final concentration of 250 nanomolar.
Then aspirate the medium from the cells and replace it with 10 milliliters of D10 plus doxorubicin. Incubate the cells in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen for exactly 24 hours. Then aspirate the medium and use 10 milliliters of D10 to carefully wash the cells once.
Incubate the cells in 10 milliliters of D10 for another six days replacing the medium regularly approximately every three days. To carry out beta-galactosidase staining, for each sample seed one times 10 to the fourth cells in at least one well of a 24-well plate containing 500 microliters of D10 so that the cells are sparse. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen overnight.
The following day, use 500 microliters of PBS to wash the cells two times. Then add 500 microliters per well of 2%formaldehyde plus 0.2%glutaraldehyde in PBS and incubate the cells at room temperature for three to five minutes to fix them. Then use 500 microliters of PBS to wash the cells twice.
Next, after preparing fresh staining solution, add 500 microliters per well and use parafilm to seal the plate to avoid evaporation which can cause crystals to form. Incubate the cells in the dark at 37 degrees Celsius in a dry incubator without carbon dioxide for 12 to 16 hours. Then use 500 microliters of PBS to wash the cells twice.
Check the cells under a normal light microscope to assess the results where positive cells will appear with blue paranuclear staining. To facilitate visualization and quantification of individual cells, use DAPI as a counterstain. Then using a fluorescent microscope, determine the percentage of positive cells versus total number of cells taking multiple pictures of the same sample to evaluate at least 100 single cells.
For each sample or condition, place one coverslip per well into a 24-well plate. Seed one times 10 to the fourth cells with 500 microliters of D10 per well so that the cells are sparse. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen overnight.
Prepare enough of a 20 micromolar EdU solution and D10 so that each sample receives 250 microliters. Remove half of the medium from each well being treated and replace it with EdU D10 solution. The final EdU concentration is 10 micromolar.
Incubate the samples at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen for 18 to 24 hours. Use the same incubation time for control and senescent cells. After the incubation, use 500 microliters of PBS to wash the cells twice.
Then add 500 microliters of 4%formaldehyde in PBS and incubate the cells for 10 minutes to fix them. Following fixation, add 500 microliters of 100 millimolar Tris pH 7.6 to the samples and incubate them for five minutes. Next, add 500 microliters of 0.1%Triton X-100 in PBS to the cells and permeabilize them for 10 minutes.
Then use PBS to wash the samples three times. Prepare 50 microliters of label mix for each coverslip, adding the components in the following order, 44.45 microliters of PBS, 0.5 microliters of copper(II)sulfate, 0.05 microliters of sulfo-cy3-azide, and five microliters of sodium ascorbate. Pipette 50 microliters of the label mix on a piece of parafilm.
Then using tweezers and a needle, lift the coverslip and rest it cell side down on top of the label mix. Ensure that there are no bubbles and that the whole surface of the coverslip is touching the label mix. Incubate the sample in the dark for 30 minutes.
Put the cells back in the wells of the 24-well plate and use PBS to wash them three times. Then use mounting medium with DAPI to mount the samples and let them dry overnight. To begin, seed seven times 10 to the fifth viable primary fibroblasts with a low Population Doubling or.
Image at least 100 cells per condition. Finally, quantify the percentage of cells that incorporated EdU by using the following formula. Carry out gene and protein expression studies according to the text protocol.
Shown here are representative images of SA-beta-gal staining in proliferating versus senescent primary fibroblasts. Senescent cells look enlarged within a regular cell body. As mentioned, it can be hard to distinguish individual cells.
Therefore, co-staining with DAPI facilitates visualization and cell counting. In this experiment, suflo-cy3-azide was used to perform copper catalyzed azide-alkyne cycloaddition to population. If the coupling of the azide to the alkyne has taken place, cells will display fluorescence under a cy3 filter distinguishing proliferating from non-proliferating cells.
Once mastered, this technique can be done in 36 hours if performed properly. While attempting this procedure, it is important to remember to be as quick as possible while handling cells outside the incubator as oscillations in the oxygen concentration might affect their results. Following this procedure, other methods like cytokine array or Western blot can be performed in order to answer additional questions like the levels of secreted proteins or understanding specific signaling pathways.
After its development, this technique paved the way for researchers in the aging and cancer field to explore how senescent cells are involved in physiological and pathological conditions in mammals. After watching this video, you should have a good understanding on how to reproducibly induce senescence in primary cells and how to assess their senescence status.
