The overall goal of this procedure is to quantify cell senescence in a rapid and sensitive way. Here we demonstrate the utility of this method in screening potential chemotherapeutic drugs. First to induce cellular senescence.
The chemotherapeutic drug doxorubicin is added to cancer cells. Next Bain A one is applied to neutralize the acidic pH of cellular lysosomes. Then cell permeate beta galacto substrate C 12 FDG is added upon cellular uptake.
The C 12 FDG is hydrolyzed and fluoresces. Since beta GCSEs is enriched in lysosomes of senescent cells, they become more fluorescent than non senescent cells. To quantify the difference in fluorescence, the cells are run on a flow Cytometer analysis of the resulting data allows for quantification of the percentage of senescent cancer cells under different treatment conditions.
The main advantage of this technique over existing methods like the beta galactose does, is say, is that it facilitates rapid and sensitive quantification of senescent cells. In fact, we first add the idea for this methods when we experience difficulty with quantifying senescent cells using the commonly used beta galactose asay. Although we use this technique to detect senescence on cancer cells, it can also be used on normal cells.
This method can help answer quick questions in the cancer field, such as whether a chemotherapeutic drug induced cancer cells to sse, which might promote malignancy. Thus, this technique could ultimately help notify new therapeutic treatments for cancer. Working in a chemical fume hood dissolved the C 12 FDG powder in DMSO to a final concentration of 20 millimolar aliquot into 0.5 milliliter einor tubes and store at minus 80 degrees Celsius.
DMSO should be used with appropriate safety conditions, also dissolve. CIN A one powder in DMSO to a final concentration of 0.1. Millimolar Eloqua the solution into 0.5 milliliter micro centrifuge tubes and store them at minus 20 degrees Celsius.
Note, the CIN should be used with appropriate safety precautions. This procedure can be performed using either adherent or non-adherent cells. Here the procedure is demonstrated using the human myeloma cell line RPMI 8 2 2 6.
These cells grow in suspension, cultivate RPMI 8 2 2 6 cells in medium containing 10%FBS and 1%antibiotics at 37 degrees Celsius and 5%carbon dioxide 24 hours prior to performing the experiment seed the cells in six well plates so that they will be in the log phase of the cellular proliferation curve. At the time of the experiment to determine the percentage of senescent cells, three cell samples will be required. Untreated and non-ST stained cells, untreated cells stained with C 12 FDG, and treated cells stained with C 12 FTG.
For each condition, a minimum of five times 10 to the fifth cells are required for flow cytometry induced senescence of cancer cells. By adding the chemotherapeutic drug doxorubicin, drug concentration and duration of the treatment should be adapted to the cell type. Tested for rpmi 8 2 2 6 cells.
Senescence can be induced by treating 10th of the six cells per milliliter with 15 nanomolar doxorubicin for 48 hours. Do not forget to include untreated sample as a negative control incubate at 37 degrees Celsius and 5%carbon dioxide. Treatment with chemotherapeutic drugs may lead to cell apoptosis.
Therefore, after two days, check cell viability by trian blue exclusion using a hemo, cytometer and a microscope. If the percentage of viability is less than 70%remove the dead cells using an appropriate kit such as the dead cell removal kit from Milton E to ensure that dead cells will not bias the subsequent analysis. Otherwise, remove the culture medium by spinning the cells down at 300 GS for five minutes.
Then after removing the supernatant at Prewarm fresh culture medium containing 100 anom or cin, A one to neutralize the acidic pH of lysosomes incubate for one hour at 37 degrees Celsius and 5%carbon dioxide dissolve the beta galacto a substrate C 12 FDG in pre-war fresh culture. Medium to a final concentration of two millimolar. Following the incubation with beil mycin.
Add C 12 FDG directly to the cells and medium to a final concentration of 33. Micromolar incubate for two hours at 37 degrees Celsius and 5%carbon dioxide. After the incubation centrifuge the cells at 300 Gs for five minutes at four degrees Celsius.
After the spin, wash the cells twice with PBS spinning down each time as before and removing the supernatant after the second wash. Resus suspend the cell pellet in 500 microliters of cold PBS then perform a co immuno labeling to further characterize the population of senescent cells. If you do not have experience with flow cytometry, the most difficult aspect of this procedure will be performing the flow cytometry analysis.
To ensure success, be sure to include the appropriate controls and seek assistance from unexperienced person as needed. Prior to running the first sample, prepare a two parameter graphic displaying forward scatter channel or FSC versus side scatter channel SSC. Then run the first sample containing unlabeled cells.
Set the photomultiplier tubes or PMTs and define a region of interest, excluding dead cells and cellular debris that might have appeared during the sample preparation gate. This region of interest in a one parameter histogram displaying the fluorescence of C 12 FDG as FL one on the log scale of the x axis and the number of events in the Y axis. The total number of events represents the number of cells analyzed.
Set the PMT so that unlabeled cells appear in the first decade on the log scale of the x axis. Determine the autofluorescence of the cells if necessary by running the unlabeled cells in the flow. Cytometer run the second sample containing untreated cells on the one parameter histogram displaying FL one place the cursor after the dimly labeled population.
This threshold will allow the discrimination between non senescent cells, which are dimly labeled and senescent cells, which are brightly labeled. Run the third sample containing treated cells. Brightly labeled senescent cells appear above the threshold determined in the previous step.
Evaluate the percentage of senescent cells by dividing the number of bright events by the total number of events if needed. The activity of the beta galacto tase can also be estimated using the mean fluorescence intensity to evaluate the senescence induced by a chemotherapeutic procedure. The commercially available multiple myeloma cell line R RRP I 8 2 2 6 was treated for two days with doxorubicin, a chemotherapeutic drug and assay for senescence as described in this video, these histograms show the fluorescence of untreated cells on the left and treated cells on the right.
A population of brightly labeled cells representing senescent cells appears in region V.In parallel, the same cell sample was stained using the beta galacto sase assay. In this representative picture entirely blue cells and partially blue cells are shown to illustrate the difficulty in distinguishing truly blue senescent cells. To illustrate the specificity of C 12 FDG in the determination of the percentage of senescent cells, the cells were also treated with various concentrations of doxorubicin.
The percentage of senescent cells was determined by dividing the number of bright events. Per the total number of events, the percentages of senescent cells were 0.8%15.78%and 26.31%for a dose of 5 25 and 50 nanomolar doxorubicin respectively. This demonstrates that C 12 FTG labeling is specific to senescent cells.
After watching this video, you should have a good understanding of how to quantify senescent cells can series or not by flow cytometry following this procedure of methods like immuno labeling be performed in order to characterize senescent cells. Don't forget that working with DMSO or cancer cells can be extremely hazardous. Precautions should always be taken while performing this procedure.
