They are primary cultures of rat dermal papilla cells. The photo includes both healthy cells (left) and dying cells (right), to show the difference between them. It's strange because they are dying flat, it seems to me like if the nuclei started to compact the chromatin and shrink, creating this empty ring between the cytoplasm and the nucleus (bottom right cells), and I really don't know if it corresponds to later stages of the process, but I can also find a lot of this flat cells completely lacking the nucleus leaving an empty space surrounded by the cytoplasm (top right cells). They start as patches of cell death and then spreads to the whole plate. I'd appreciate any clues, I haven´t seen this before! Thanks!!!
The picture of the dying cells you are showing illustrates nuclear chromatin compaction and is a late part in the apoptotic process. In early apoptosis the cells appear "normal" in light microscopy - early apoptosis starts with descrete membranal blebbing and chromatin packing towards the nuclear membrane that can only be observed in transmission electron microscopy.
I completely agree with the previous suggestions but want also to make you aware of that you are culturing primary cells from dermal papilla cells - you have to consider the normal physiological process that occurs in skin not only rat but any, also human - dermatocytes/keratinocytes undergo a physiological gradual apoptotic process as they move from the basal membrane up-wards to contribute to the formation of the completely keratinized superficial layer of the dermis (skin). If you can rule out that this is not mucoplasma or some other environmental signal in your culture causing apoptosis, you must consider that it must be a normal physiological behaviour of the cells of your primary culture - they are simply following their internal programming being skin cells. In this situation, to prevent them from going to apoptosis you must arrest them at a certain developmental stage and immortalise them.
Hey Elisa,
I'm not an expert on cell death but I've recently learned that keratinocytes (cells of the epidermis) undergoing apoptosis following irradiation with UVB (so-called 'sunburn cells') show the very same morphology including this nuclear halo, which, as you said, likely results from chromatin compaction.
Have you tried staining your cells for apoptosis e.g. doing a TUNEL-Assay or a cleaved caspase 3 staining?
Check for Mycoplasma infection! Had it ones and cells looked exactly the same as your cells.
The picture of the dying cells you are showing illustrates nuclear chromatin compaction and is a late part in the apoptotic process. In early apoptosis the cells appear "normal" in light microscopy - early apoptosis starts with descrete membranal blebbing and chromatin packing towards the nuclear membrane that can only be observed in transmission electron microscopy.
I completely agree with the previous suggestions but want also to make you aware of that you are culturing primary cells from dermal papilla cells - you have to consider the normal physiological process that occurs in skin not only rat but any, also human - dermatocytes/keratinocytes undergo a physiological gradual apoptotic process as they move from the basal membrane up-wards to contribute to the formation of the completely keratinized superficial layer of the dermis (skin). If you can rule out that this is not mucoplasma or some other environmental signal in your culture causing apoptosis, you must consider that it must be a normal physiological behaviour of the cells of your primary culture - they are simply following their internal programming being skin cells. In this situation, to prevent them from going to apoptosis you must arrest them at a certain developmental stage and immortalise them.
Good point, Lucia!
When culturing keratinocytes for instance, the medium needs to be devoid of calcium to prevent differentiation, since in intact skin there is a gradient of calcium present across the epidermis which 'tells/allows' the basal keratinocytes to progress to a more differentiated state. Depending on your research question, perhaps the depletion of calcium from the culture medium could be an option here, too.
It looks like a heavy Mycoplasma infection, small organisms both intra- and intercellular.
Hi Elisa!
Can you use viability stains to know if the different cells are alive or a cell type? if dying, the intact shape suggests necrosis. Good luck and hope you trouble shoot this!
this can be due to some contamination or mycoplasma, please check your culture by using mycoplasma detection kit.
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