Can we distinguish the normal and senescent fibroblast in culture from their morphology? If during the prcess of trypsinisation if they are not getting trypsinised then can these fibroblast be said to be showing sign of senescence?
Because senescent cells are bigger and flatter, they may well take longer to detach for that reason - simply time for the EDTA and trypsin to diffuse underneath the cell. It is not impossible that they are also better attached, because there are definitely cytoskeletal changes, but I don't know of data on that.
If you want some markers of senescence to quantitate the effect of your chemical, you can use beta-galactosidase or p21 or p16 or a DNA damage marker (if doing immunostaining for that, look for tiny foci inside the nucleus). Best to check more than one marker, as beta-gal is not fully specific and the others vary with different cell types and treatments etc.
Hi Ravindra, I am assuming that you are using primary cells and not a tissue culture cell line, right?
It has been awhile, but I used to work with primary chicken embryo fibroblast cultures and when you saw "bubbles" or "vacuoles" (non scientific terms) in the fibroblasts using a regular microscope, they were too old to infect with Rous Sarcoma virus and hope to get transformed cells that could proliferate.
Even if you got early passage fibroblasts to infect, the transformed cells were not immortal. They had a finite passage number and when you saw bubbles or vacuoles in the cells, they were too old to do any experiments with.
Compare panels A and B in the following link
http://mcb.asm.org/content/18/3/1611/F2.expansion
Thank you Steingrimur and Goodwin for your suggestions. Yes we are using primary cells. Actually we wanted to test one chemical if that is inducing the senescence in the fibroblast cells. And the treated cells are not getting trypsinised even after the trypsinisation for 7minutes. But untreated cells are getting trypsinised just in 30 seconds. So I was wondering if senescence fibroblast have srong attachment as compared to the non senescent fibroblast.
Because senescent cells are bigger and flatter, they may well take longer to detach for that reason - simply time for the EDTA and trypsin to diffuse underneath the cell. It is not impossible that they are also better attached, because there are definitely cytoskeletal changes, but I don't know of data on that.
If you want some markers of senescence to quantitate the effect of your chemical, you can use beta-galactosidase or p21 or p16 or a DNA damage marker (if doing immunostaining for that, look for tiny foci inside the nucleus). Best to check more than one marker, as beta-gal is not fully specific and the others vary with different cell types and treatments etc.
We stained the same treated fibroblast with the DAPI and it is observed that in those cells which were not getting detached by trypsinisation are without nucleus(not getting stained with DAPI). But there morphology is quite fine well spread. Can such fibroblast be exist in the culture which are without nuclei inside? They are showing there morhology like as images shown bellow.
yah under phase contrast we can see they have prominent nuclei but those nuclei are not stained with DAPI.
Hi Ravindra, looking your picture brings back not very fond memories.
When cultures of primary chicken embryo fibroblasts got old, they had that appearance. Cytoplasmic spots (vacuoles) and a bloated nucleus.
BTW, how do you post images onto RG?
Any chance you could show us a DAPI image of that? I have never seen cells that have visible nuclei but the nuclei are not stained by DAPI. In your image the nuclei are the pale oval areas (one cell has 2 nuclei, which is quite common in senescent cells, 2 or more..) and the dark spots inside the nuclei are the nucleoli. The nucleoli are not stained by DAPI because they are mainly RNA. Prominent nucleoli are also a common feature of senescent cells.
DAPI is not cell permeable if you used live cells. DAPI required cell permeabilization in the presence of citrate for DNA staining.
Try Hoechst33342 in live cells in medium and incubate for 20 mins to stain nuclei. Hoechst 33342 is cell permeable
@Steingrimur yes we can just attach a file with the link after the massage box,only one thing one need to mind is the image format should be in jpeg not tiff.
@Dorothy, we don't have phase images with DAPI staining. But there are some images when we saw them under high fluroscence intensity it looks like the their DNA is dispersed all over in the cytoplasm. I am attaching the same images in two different conditions.
That seems interesting.
1. It could be RNAs all over cytoplasm (Check with RNAse treatment followed by DNA staining to rule out RNA in cytoplasm.
2. It could be DNA seeping into cytoplasm (Check Lamin A/C integrity by Western Blotting: Lamin A/C helps anchor DNA inside nucleus)
3. It could be mitochondrial DNA exposed into cytoplasm (Try immunofluorescence in these cells with mitochondrial matrix proteins +DAPI)
4. It could be mycoplasma accumulated in cells by phagocytosis (wish this is not the case: A few dots outside cells raises this possibility but the size seems larger and hence it might not be the case).
Hi again,
Most of what you show is just the large nuclei of the senescent cells. In the cytoplasm of some cells there are a few small blobs of DNA. These may well come from other cells that have died by apoptosis, where the nucleus breaks up into just such small blobs. The fragments of apoptotic cells are commonly phagocytosed by other cells, and I'd guess this is what those smaller blobs are.
The nuclei also show some evidence of SAHFs - senescence-associated heterochromatic foci. This is when the nucleus of a senescent cell forms a number of foci of heterochromatin (but it is still within the nucleus). This is seen in fibroblasts at senescence, but not all cell types. First reported by Narita M et al - you can see pictures in their paper here:
Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Narita M, Nũnez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW. Cell. 2003 Jun 13;113(6):703-16.
PS - actually looking at the second picture I agree the cytoplasm seems to be more clearly stained (as well as the nucleus). This does tend to indicate mycoplasma, regrettably. If you check at higher magnification, mycoplasma look like many tiny dots in the cytoplasm.
http://www.ncbi.nlm.nih.gov/pubmed/12809602
Thanks a lot Dorothy and Goodwin for your valuable suggestions. :)
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