Yes, longer than necessary serum-starvation can cause apoptosis - especially in tumor cells.

Also, even though a tumor cell line looks fine after serum-starvation, RE-stimulation with serum can also induce apoptosis. This can be a bigger problem !

You are highly recommend to do a time course of the effect of low serum on your cell type. Try times of 4hr, 8hr, 12hrs, 24hrs. Do an apoptosis assay at each point to see what happens. Also, if you find a time point that is satisfactory, try a 24hr RE-stimulation with serum and see if the cells look OK>

Good luck !

Yes, longer than necessary serum-starvation can cause apoptosis - especially in tumor cells.

Also, even though a tumor cell line looks fine after serum-starvation, RE-stimulation with serum can also induce apoptosis. This can be a bigger problem !

You are highly recommend to do a time course of the effect of low serum on your cell type. Try times of 4hr, 8hr, 12hrs, 24hrs. Do an apoptosis assay at each point to see what happens. Also, if you find a time point that is satisfactory, try a 24hr RE-stimulation with serum and see if the cells look OK>

Good luck !

Serum starvation normally halts the cell cycle at the G0 stage. But in case of prolonged arrest, cells of ALL (since they are fast growing cells of a cancer type), may undergo apoptosis. The best way to find out the time point is to do an assay with time dependent study of apoptosis like Prem has suggested.

But I think this will be a critical thing to do because if you serum starve the cells for way too long, they will surely die in culture. But the 'way too long' has to be determined by you.

As for increase in the apoptotic protein expression, I would just collect an aliquot of cells (since it is a suspension, you can afford to do that) at different time points and lyse them. At the end of the experiment I would perform a western blot of the collected cell lysates to analyze what I have in hand.

Many thanks of you

Firstly to answer exactly to your question of serum starvation for suspended cells -

a) Spin the suspension cell culture and collect the pellet while discarding the supernatant.

b) Wash the cells once with PBS and discard the supernatant

c) resuspend the washed pellet into fresh culture medium with out Serum

d) count the cells and start the experiment

e) set different time intervals such as 0 hrs, 6 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs and so on

f) harvest the cells at the end of each time point

g) prepare cell lysate from the harvested cells if you want to analyze by western blotting

HOPE THIS ANSWERS YOUR QUERY

Yes, of course.

You should do a titration of serum starvation and a time course.

In my opinion after washing once with plain media and re-suspending cells using a 0.01% to 0.05% FBS containing media with 1X PenStrep should hold the cells for up to 72 hours and almost 99% arrest in the cell cycle. Depending on cell type you can also use horse serum in the same 0.01-1.0% range.

thank you all, I tried based on titration, I think that 12 hours incubation is the best time for serum starvation.

The above suggestions are all useful. However, the result will be that for a population of cells possibly in various stages of apoptosis.

You may also want to use Annexin V with Propidium Iodide (PI) to count early stage apoptotic cells to correlate with your protein sample data. This could be done with either flow cytometry or fluorescence microscopy (former is easier). The Annexin V measures exteriorized phosphatidyl serine (a very early step in apoptosis) while PI measures DNA if the cell membrane is not intact. Apoptotic cells will be Annexin V positive and PI negative (intact membrane). Necrotic cells will be positive for both. Healthy cells will be negative for both. The amount of your apoptotic protein is likely to vary with stage of apoptosis, so this comparison may be useful, if not critical. Your Western blot will only tell you how much protein is in your population but the Annexin V and PI assay will give you per cell data as to how many are apoptotic.

@ Christopher Lange ; many thanks of you, I used Annexin V and cell cycle PI

Is there a way to sort out the Annexin stained cells since some cells may be high for Annexin and others low depending on the state of apoptosis. I was wondering if the protein in question has any expression changes pre or post apoptosis. Sorting cells and western blotting for the protein could yield more meaningful data I presume.

Flow sorting lets one collect the early apoptotic cells (annexin V positive PI negative). This is a selection for early apoptosis. The exteriorization of the phosphatidyl serines appers to be an all or nothing process, not increasing per cell once it happens. One could sort and then test for other proteins but they, e.g., caspases, usually are not present until later in apoptosis.

I think I would also keep in mind that prolonged starvation or any treatment to induce apoptosis could lead to secondary necrosis as well. Hence looking for not one but a few indicators of apotosis such as Annexin V and caspase cleavage as mentioned above and PARP cleavage which is distinct for apoptosis versus necrosis would be very useful.

I agree w Vidya but note that these different "markers" develop sequentially, so a time course study will be particularly important to a proper interpretation of the results.

I did 12h serum starvation, then harvest cells at 4h after drug& growth factor exposure.

in normal condition, percent of death was among control cells less than 3% but in serum starvation statue after 16h : 10%

is that a problem?

Maysam, You should probably do a time course study of fraction of apoptotic cells from zero to 24 or 48 hours with and without serum starvation, both without any drugs, and also the same time course with your drug exposure group(s). You can then use the same starvation duration no drug control vs the drug results (at each time) but then also check that the difference between with and without serum starvation controls is not large enough to skew the population of cells being assayed.

I think the best time period for serum starvation is 24 hours.

24-48 is safe because doubling time varies greatly in cancer cell lines.

The importance of a time-course study is that apoptotic cells may already be reproductively dead from other causes, such as mitotic death. This is seen clearly with fibroblasts (V79). When irradiated with ionizing radiation, they die a reproductive death by mitotic failure. Normally there is no apoptotic death for these cells, but a few cell cycle times after irradiation, when the vast majority have been sterilized by mitotic failure, one sees some low levels of apoptosis. So apoptosis is a mechanism of removal of viable but reproductively dead cells (i.e., they don't die until attempting mitosis) as well as a mechanism of programed cell removal.

you can prepare your media without serum and grow your cells with that media for 24 hours not more so as to find the protins that involve in apoptosis, then you can use Annexin V-CY3 assay.

Quiescence is a state where your cells should be resting and not dying. It is hard to maintain for more than 2 or 3 days and will take you some time to establish for your cell line. If you want to kill the cells by apoptosis then just removing the FCS from your culture media should kill most cells. If you want to make them quiescent and then kill them with a drug then you need to think about your culture conditions. I supplement low FCS culture with BSA, as when you remove the FCS from your media you also remove the BSA, and that does a number of things such as bind other proteins and help stabilise the membrane. I also add selenium, which whilst toxic, is needed in trace amount by cells as a co-factor in some detoxifying enzymes such as glutathione and thioredoxin reductases and Selenoprotein P which transports selenium and prevents cell membrane per oxidation. Selenoprotein P is present at high levels in plasma and transport selenium in the blood (and is therefore missing in low FCS containing media). I also add transferrin. Whilst cells need iron, it can be toxic, and transferrin acts as an iron transporter to give the cells iron in a non-toxic form. If you add these compounds to you culture media then they should come out of the cell cycle and not enter apoptosis for about 48-72 hours, but again this depends on you cell type. So if you cells are quiescent then you can add your cytotoxic drug and they will die and you should have a minimal contribution from just serum deprivation.