I'm not at all an expert on this, but I would have thought some sort of UV treatment might be a way of accelerating ageing in cells

Try BrdUrd incorporation

With regards to markers, you can follow the decrease in proliferative ability (a surrogate for senescence) by staining for Ki67 or PCNA. Not sure you can drive the cells faster if they already grow at max rate!!

Sa Beta gal it is only a quick way to look to cells to see whether they are close to senenscence but it is NOt a senescence marker. Cells can be SA beta positivi also in dependance of plate density. Senescence is defined as an irreversible non-proliferative state. Typically real senescent cells are p21waf1 positive and p19arf positive. RB is dephosphorylated and morphology is quite peculiar they look flat with irregular margins and many long pseudopods. The cytoplasm is gfull of granules the nucleus may have more then one or two nucleoli. They can stay like this almost forever. UV treatment is reported to accellerate senescence. Years ago David Beach described in CELL the prosenescence properties of protooncogene RAS. Is this of any help?

There exist several approaches to evaluate cellular markers for ageing. Cellular senescence is sometimes considered as cellular ageing. However, it is very important to know that aging is considered a progressive functional decline in organisms. Thus, to use this term in cells is quit correct. For analyzing physiological changes produced during "aging/senescent" cells, could be very useful to look for specific post-translational modifications (i.e. acetylated protein levels in mitochondria. Goto S. Ann NY Acad. Sci. 2010; ubiquitinated proteins levels in whole extracts. Li F. Mech. Ageing Devel 2008). However, the analysis of post-translational modifications in histones may be very useful for this pourpouse. In fact, decreased levels in H3K9me3 and H3K27me3 has been observed in cellular models and fibroblasts from patients with progeroid syndromes (Shumaker D.A. PNAS 2006) and also increased levels of ubiquitinated H2A/H2B. Marimoto S. Arch Geront Geriat. 1993).

You can also measure the telomere length by measuring by PCR (T/S ratio) or using shouthern blot (Roche Kit).

I hope that this info can answer your question.

Dear all,

thank you very much for your valuable input and hints at relevant methods and publications. Let's keep the fingers crossed that my gene of interest is indeed relevant for longevity. Any further thoughts and ideas are warmly welcomed.

Here are the lists of methods that you may use for cell senescence assessment.

1. Cell morphology: cell flattened and enlargement, irregular

arrangement, accumulation of granular cytoplasmic inclusions, reduced saturation

population density, and it's easy to be observed under light microscope.

2. β-galactosidase expression by β-gal staining: as you mentioned

3. Cell cycle assay by flow cytometry: see the ratio of cell cycle phases

4.Senescence-associated molecular markers by western blotting: such as anti-p53 (but this marker is also the indicator of apoptosis), anti-p16INK4, anti-p21Waf1, anti-CDK4, anti-CDK6

5. The expression and phosphorylation status of the RB protein by western blotting

6. Up-regulated genes expressions by qPCR: collagenase-1, apolipoprotein J, fibronectin, α1(I)-procollagen, osteonectin, SM-22, SS-9

Hope this would help more or less :)

All the best!

To increase the senescence of in vitro cultured cells (cells that are not transformed), you can try either:

1. to culture the cells in rich mediums with high glucose content.

2.always subculture the cells when they are at very high confluence (90~100%, very crowded)

see if this could help

Alexander, Are you sure HEK is not immortal? It seems it is long established cell line. 64 chromosomes of average, suspension culture, transformed.... Sure cells can stop dividing for many reasons related to culture stress or better say sub-optical conditions. I guess you need to define more what you trying to study. Speculatively, your culture conditions are sub-optimal for this cell line and your gene or transformation per se (random integration in first experiment) allowed you to select a cells with survival advantage.

Hello Andrei,

all valid criticism.

A few clarifications, just to avoid misunderstandings:

HEK293 is an adherent cell line but with low attachment to the surface. They can be easily flushed off with a pipette. In some protein production experiments, they can be tricked into suspension cell culture as to increase protein yield. In my case, I used them as adherent cells.

I used DMEM + high glucose (4.5g/l) + GlutaMax + 10% FBS + 1% PenStrep as medium for all flasks in the first round of experiments. cosy 37°C, 5% CO2 in humid atmosphere. So, equal treatment for all cells in the same incubator. If you can spot sub-optimal conditions, I would be happy for you to do so.

293 cells were transformed with an adenovirus, while 293T cells were 293 cells immortalised with the large T antigen and are resistant to neomycin.

I certainly used 293 cells, as the non-transfected cells were sensitive to G418.

Random integration of the insert: certainly correct.

Alexander, I have needed to mention that I do not have experience with HEK293. I made small search. Here is very interesting discussion and caution on the use of HEK293 as a model: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224126/ where these cells are called immortalized but with delayed senescence (hm, kind of confusing statement :)).

You can contact your provider about optimal medium and culture procedures. You can also check some published papers of the culture of these cells. Additionally, not presuming anything ok?, suboptimal culture can originate from expired medium, not checked CO2 % in incubator (they show 5% but actual % may be different), long handling outside incubator , micoplasm contamination, not heat inactivated serum et cetera. (they also mention that cells shall be passaged before reaching 90% confluence, where 90% confluence is very arbitrary to my experience).

I agree with Barbara Ridolfi too, take a primary cell line, like normal dermal fibroblasts for senescence studies. Good luck!

Hello Andrei,

thanks for following that one.

After all, HEK293 appears to be a suboptimal model for my question, as 293 are initiated owing to their supposed immortalisation but not malignant yet.

Yes, I agree that primary cells would be the best model to follow up senescence.

I also agree that the 90% cut-off for splitting is quite arbitrary (why not 79.3%, 88.5%, or 94.6%?) but appears to be one of these rules of thump that work more or less well and people are intrinsically inert to change a running system or validate the system, as it costs time and efforts.

Thanks

Gamma irridiation is the most effective way to induce cell senescence in culture.

Alexander, what is the question you are pursuing? If you are looking for replicative senescence, I do not think HEK293 are the choice. The senescence-like phenotype you observed in your cells may be due to the stresses your overexpressing cells are exposed to, which I do not see it as a feature of aging.

If you are looking for premature senescence, which might better suit the case, beside SA-beta-gal, you may look for the cell cycle distribution, BrdU incorporation and DNA damage, the presence of heterochromatin. There is no definite assay for cell senescence, unfortunately.

For accelerating cell senescence (aging), you can impose various stresses according to your case, like oxidative stress(H2O2), DNA damage imposed by gamma irradiation and so on.

Ionizing irradiation is an aging stressor. Telomere length and p16INK4a status are good biomarkers of aging.

As a general concept the inhibition of Sirtuin activity accellerates cellular senescence. Sirtinol in fact has been reported as able to do so.

As to plants, leaf mitochondrial superoxide dismutase (SOD) is induced during senescence. You can check this easily in gel or by measuring enzymatic activity in the spectrophotometer. I suppose that human SODs are also altered during senescence. Protein carbonylation and lipid peroxidation are also good markers of ageing.