Hello,

This phenotype is very similar when actin cytoskeleton is shrinked ! This do not definetely imply that cells undergo apoptosis albeit they will after a prolong exposure. Did you try to change the medium and take off etoposide to see whether they could restore their shape. Normally a cell that are deeply engage into the apoptosis should not rescue from it (unless for very early time point).

Best regards

Daniel

This is the control group.

They look as if they were treated with staurosporine. Maybe the chemotherapeutic agent works in a similar manner like staursporine. Best is to measure cell death and then you know, might be that they are dead.

They are MG63

OS, this is osteosarcoma...

And treated with etoposide

Apparently, the cells where really induced to apoptosis. If they are less abundant, that mean that the other cells are detached in the culture medium... They look on there way to dye, stretching very hard on the focal adhesion points, everything normal indeed. You should take the supernatant a part, trypsinized you cells and resuspend them with the supernatant to perform a viability assay and/or a test of clonogenicity is you don't have access to a flow cytometer. If yes, a staining with DiOC6(3) 20 nM (10 min at 37°C) versus PI (2microM/ml) with give you te idea of their plasma membrane permeability and I bet on t e fact that they have decreased mitochondrial membrane potential. Many other staining are possible.... I am ready to tell more if needed

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PS: Better tell every one what is the inducer.... from th ebeginning.

So, etoposide being the one, the cells are on their way to die....

72 h is a bit long for such experiments...

As per my observation the concentration which you against these cells is not sufficient.Please try with more concentration.

it seems that your cells are becoming senescent, which could explain the decrease in proliferation that you observe, a staining of SA-b-galactosidase could confirm or refute this hypothesis

Indeed, Etoposide is really toxic (I use 10 micromolar O/N as a control of apoptosis determined by Annexin V/ PI and Facs) as it is an inhibitor of Topoisomerase II and it generates DNA double strand breaks. I do not not know the stability of this agent in the culture medium. At 72h, the remaining cells could be surviving cells that have repair their DNA....This is only a suggestion of course..I found the old paper below

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Cancer Chemother Pharmacol. 1991;27(5):354-60.

Instability of the anticancer agent etoposide under in vitro culture conditions.

Mader RM, Steger GG, Moser K, Rainer H, Krenmayr P, Dittrich C.

SourceDepartment of Chemotherapy, University of Vienna, Austria.

Abstract

Degradation of etoposide is rapid under in vitro culture conditions. At pH 7.4 and 37 degrees C, the isomerisation of trans-etoposide to the inactive compound cis-etoposide has a half-life of 2 days in Dulbecco's modified Eagle's medium and results in the loss of 90% of the active drug within 1 week. As a consequence, prolonged incubations with etoposide in in vitro assays may lead to erroneous interpretations ignoring the real in vitro situation. The degradation is not influenced by organic compounds such as bovine serum albumin or amino acids but depends strongly on the pH value and, to a lesser degree, on the ionic strength of the medium. Therefore, we propose a mathematical correction based on the pH value so as to obtain the real exposure of cells to trans-etoposide during in vitro assays.

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Best of luck, Claudie

In my opinion they are dettaching. I agree with Patrice Petit

Hello,

This phenotype is very similar when actin cytoskeleton is shrinked ! This do not definetely imply that cells undergo apoptosis albeit they will after a prolong exposure. Did you try to change the medium and take off etoposide to see whether they could restore their shape. Normally a cell that are deeply engage into the apoptosis should not rescue from it (unless for very early time point).

Best regards

Daniel

DNA damage by etoposide also trips cell cycle check points that halt cell division. As others have said, most of the apoptotic cells may have disappeared by 72 hours post-treatment. Some of the remaining cells may have rounded up to divide but then triggered the mitotic check point. The best way to know for sure is time-lapse microscopy if you have access to the equipment.

I dont think this is senescence. Senescent osteosarcoma cells and normal osteoblast have a very distinct flattened morphology once they enter senescence. At looks almost the opposite than what you see here. I would also bet that your cells undergo apoptosis, but since they are a cancer cell line, it might be they dont show all the common features of a typical apoptotic cell. But they definitely look very much stressed. I would stain with Hoechst to check for nuclear condensation, and do a selective extraction of fragmented DNA (including the floating cells, as Patrice Petit pointed out), which you might run on a gel to see the typical DNA ladder of apoptosis. Since these two features are early apoptosis markers, they are more reliable than the late morphological changes.

By the way, do you see membrane blebbing if you view the cells with higher magnification ?

I can't speak to whether your etoposide concentration is optimal for inducing cell death, but the morphology is very reminiscent of a G2/M block.

A simple typan blue staining will tell you whether their plasma membrane has changed permiability. To tell whether they are induced to apoptosis, do the classic Annexin V/PI staining and flow cytometry.

sometimes this feature may be due to mycoplasma or adherent bacterial contamination. If not, then cells cytoskeleton has disturbed and cells will die with time......and it may be necrotic or apoptosis but not senescence. Cell number is less because dead cells would have detached and floating ...if not......then just check if cells are engulfing adjacent cells........its rare case.

Hi

I assume that you are using etoposide in this study-can I ask what the cell line is? Also when you say "in a previous study", do you mean in the same cell line or in a different one?

I would suggest that they are dying, but that they have the wrong morphology for senescence (too small). The main question I would suggest is is it necrosis or is it apoptosis. sometime when you use very high doses of chemotherapeutics the cells die by necrosis rather than apoptosis, and often necrosis takes a lot longer that apoptosis (it does not always happen though).

If you have access to a flow cytometer the I would do annexin V staining to determine apoptosis vs necrosis as suggested, but you could also just do a simple PI or dapi stain to look at the nuclei; apoptotic cells undergo characteristic condensation, whilst necrotic nuclei swell up and then fragment, even if the cells shrink..

I would discount the mycoplasma if the stock cells are growing normally and look OK- etoposide is very toxic!

Hi.

You need to reconsider stimulation's concentration and time duration. I would suggest induce cells by concentration dynamic and time dynamic . Example if your chemotherapeutic agent is Etopoxide , you can try 50, 100, 200, 500 mkM for 24h (depends on your research direction , for cell death high concentration , for DNA damage/repair /recovery may use low concentration 10,20,50 etc ) . I also agree Andrew Sunters suggestion. DAPI stain is very easy method to see apoptotic nuclear, chromatin condensation etc

Good luck

Greetings.

Tina, as to morphological changes concern, Daniel Bouvard is correct: your treated cells were experienced a stress that was accompanied by constriction of cytoskeletal microfilaments and loss of focal adhesions. This is a common response of adherent cells to different “stressogen” factors, especially those, which induce oxidative stress. See an example on another cell lineage at:

Gorbunov NV, Atkins JL, Gurusamy N, Pitt BR. Iron-induced remodeling in cultured rat pulmonary artery endothelial cells. Biometals. 2012 25:203-17.

OK for the high level of stress! They look as dying cells. I think also that DAPI would be informative.

In my opinion, attempting to cause apoptosis I think you selected a tough group that changed their phenotype and genotype probably something else.............

I agree with prior comments. Staining to determine whether their is condensation of nucleus, fragmentation whether in the dish (on coverslips) by DAPI staining or running on gel (compare to supernatant DNA) would be useful. These cells may be stressed or may be a population of resistant cells? What happens if you remove etoposide and replace with fresh medium? Do they start growing again? Change phenotype?

I agree to a lot of valuable comments given above. My comments are most likely undergoing apoptosis as seen by their morphological changes. Apoptosis by definition is genetically detemined physiolological process of cell death characterized by specific morrphological changes such as membrane changes( blebbing), shrinkage of the cell size, condensation of chromatin, fragmentation of DNA and fragmentation of the cell leading to apoptostic bodies. Since these are of mesenchymal morphology cells there appears to be shrinkage the rest has to be detected by using specific apoptotic assays from eg. 'Life technologies' or other companies that offer apoptosis detection kits eg. ANNEXIN -PI, TUNEL Assay, then detection of fragmentation by detecting the DNA ladder, just to mention some. You can measure apoptosis quantitatively using Flow cytometric assays or morphological changes may be scored by even microsopy following staining. Prior to all this it is important to doa dose response and a time course. Membrance changes are detected early and fragmentation the last and taking several time points of treatment of the agent and several doses of the agent is vital for understanding the drug and comparing with your control to see its effect, in other word performing a dose response and time course of the experiment will provide a thorough understanding of your experiment.

Thank you for all of your comments, I have taken into account a lot of what you have said. Next week will be another experiment. It would be interesting to see if the growth/morphology changes by removing etoposide and changing the medium.This week the cells were treated with etoposide for only 48 hours and stained with DAPI (Top; Control, Bottom; Etoposide).

Hi Tina, to visualize nuclear morphology using DAPI or Hoechst staining, set the microscope to a higher magnification (40 x lens), switch of the normal brightfield illumination (fluorescence only) and removed the phase-contrast filter.

regards Michael