Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.
We describe here for the first time a method that allows high-throughput drug screening for senotherapeutic compounds that have been shown to be beneficial in aging-related diseases. In fact, several clinical trials with drugs tested in this essence are currently ongoing. The main advantage of this technique is to distinguish between two types of senotherapeutic drugs, senolytics and senomorphics, by using the well-established detection of SA-beta-Galactosidase in senescent cells.
This technique is an important screening tool for novel senotherapeutic compounds. In fact, several compounds that have been identified with the help of this screen are featured in various high-profile publications, and are currently in clinical trial. This technique is extremely versatile, and may be adapted to several high-throughput screens.
In fact, it's possible to multiplex screens, and screen for compounds that suppress senescence via the activation or inhibition of one of several pathways. Mince each embryo into one millimeter pieces and pipe it up and down several times. Next, add 10 milliliters of growth medium to the tissues of each embryo.
Plate them in a 10 centimeter culture dish, and incubate at 37 degrees Celsius in 3%oxygen and 5%carbon dioxide for two to three days. And incubate the rest of each embryo with 0.25%trypsin EDTA mixture for 10 minutes. To trypsinize cells, first, carefully remove the growth medium and wash the cells with 10 milliliters of 1X PBS twice.
Then, add two milliliters of a 025 trypsin EDTA solution to the cells, and incubate them at 37 degrees Celsius for two to three minutes. Next, inspect the cells under a microscope to make sure that the cells are detached from the surface. Add the same amount of growth medium to terminate the trypsin digestion, and transfer the cells to a conical tube.
Then, centrifuge the cells at 200 times G for three minutes. Discard the supernatant, and add fresh growth medium to re-suspend the cells. Next, count the cells and seed them in new plates at the projected cell density.
For non-senescent subcultivation, first, split confluent cells at a one-to-four ratio. Then incubate cells at 37 degrees Celsius in 3%oxygen and 5%carbon dioxide to extend for another passage. Next, to induce cell senescence, seed split confluent cells from passage one at a ratio of one to four and incubate them at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide environment for three days.
To monitor cellular senescence, with an advanced Coulter Cell Counter system, measure the gradual increase in cell diameter and cell volume during each trypsinization step. To begin beta-Gal screening assay, seed 5, 000 senescent cells or 3, 000 non-senescent cells per well in 96 well plates. Then, add 100 microliters of growth medium to each well and incubate cells at 37 degrees Celsius and 20%oxygen and 5%carbon dioxide for six hours.
Next, add drug dilutions to MEF cells and incubate the cells at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide for 24 to 48 hours. After incubation, remove the drug solution and wash cells with 100 microliters of 1X PBS. To induce lysosomal alkalinization, pre-treat cells with 90 microliters of bafilomycin A1 solution, diluted in fresh cell culture medium at a final concentration of 100 nanomolar.
Then, incubate cells at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide environment for an hour. For fluorescence analysis of SA-beta-Gal activity, make a fresh working solution of C12FDG diluted in growth medium, at a final concentration of 100 micromolar. Then, add 10 microliters of the working solution to the culture medium at a final concentration of 10 micromolar, and incubate cells at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide for two hours.
Wash cells with 100 microliters of 1X PBS. Next, add two microliters of a 100 micrograms-per-milliliter Hoechst 33342 dye at a final concentration of two micrograms per milliliter. Incubate the cells at 37 degrees Celsius, 20%oxygen, and 5%carbon dioxide for 20 minutes.
After incubation, remove the media, add 100 microliters of fresh growth medium, and proceed to imaging. In this study, a high-content fluorescent image acquisition and analysis platform allowed for identification of senolytic drugs, such as 17-DMAG, that reduced the overall SA-beta-Gal activities in murine embryonic fibroblast cell cultures containing senescent cells in passage five. The software-generated quantitative analyses of mixtures of senescent and non-senescent cells allow for the clear demonstration of senescent cell-specific effects, and the elimination of background beta-galactosidase activity by bafilomycin A.Make sure all necessary cell controls are included, especially untreated senescent cells and senescent cells treated with a positive control.
This essay includes working with primary cells under oxidative stress, which can lead to variation that need to be monitored. This procedure is the first step in the detection of senotherapeutic drugs. Additional cell viability and cell senescent essays need to be performed to confirm the screening results.
Several new senolytic drugs have been detected using this essay, including the first senolytic drug combination dasatinib quercetin, heat shock protein 90 inhibitors, Bcl-2 family inhibitors, and the polyphenol fisetin.
