Dear Younes BOUALLEGUI

It is hard to identify the cell type with that picture.Can you add some cell culture details (cell density, culture time, used medium, oxygen levels and so on)?

I think those cells look unhappy therefore it's not enough to identify the cell type. I would call it a visual artifact due to a cell culture issue.

I used to get some cells like those while culturing mouse kidney fibroblasts. When I plated them at low density (just 25 or 50,000 cells per 10 cm dish) or when my antibiotic selection killed too many cells. They would lose the normal morphology because of the lack of contact with other cells, they were just trying to survive.

Good luck

Hi Alexandre,

Anyway thank you very much for your reply, but just to give you some more details, those cells are hemocytes (or blood cells) withdrawl from bivalves molluscs (Mytilus galloprovincialis), the cells look very happy and very comfortable at least in the best of my knowledge and the little experience that i had since i worked in the laboratory of environmental physiology of Pr. Laura canesi where i learned how to manipulate those cells, beside that, the photo is a photo of a field from a stained slide containings many fields but i choose the most less density field, because it helps in my opinion to identify the cells types,

Other thing, i mean by identifying the cell types, that from these cells which one is a granulocytes or hyalinocytes and from these two family which one is basophilic, eiosinophilic or neutrophilic and this one is the hardest thing because it depend to the colour (pink, violet dark or lilas, orange, blue or red)

As it appears from the photo that the majority of the cells are coloured by the violet colour which may vary from the light to dark level, but i have some skepticism about the existance of the light blue and the light pink, which is very important to annotate the cells sub-families (to the best of your knowledge the granulocytes type is distinguishable from the hyalinocyte type by the nucleus/cytoplasm ratio).

I hope that it's more clear now and that helps to help me,

with all my best regards!

Hi, it is difficult to me give proper suggestion looking at the Photo. Did you consider  they coud be "atypical" Haemocytes? the cells resemble "A cells" in Mytilus Disseminated Neoplasia.  Are there some mytosis around? However  If you want share some slide you can try send me one or two smears at my address: Prof. Gionata De Vico, University of Naples Federico II dept of Biology, via Mezzocannone, 8, 80134, Naples (Italy). Best regards.

Dear Younes BOUALLEGUI,

apologize for my interfering:

>  

If you don't mind my asking about telling us at least also the  >>application of stain you used (=MMG=May-Grünwald-Giemsa,which reference?, on fixed or native cultured cells?, etc.)...  Additionally I would like to say that "colors" in your image always depend on color-setup of the monitor of RG-users...  so it might be that another person only can discriminate "dark blue spots" which might be dead cells or detritus on the one hand    from predominantly ONLY violet (magenta?) stained [roundish] cellular profiles, containing intracytoplasmic vacuoles as well as some granular substrate besides a nucleus/nuclei (dark blue) in two cell profiles on the other hand.  One additional cellular profile displays a stretched, spiky appearing cellular projection (which could be interpreted at least in two ways: stress or morphology of...). 

Don't know about a good knowledge on hemocytes in bivalvia..so it might be a bit troublesome to come to the (a) right / correct result.

It would be perhaps of help if you could add also a microscopical image out of   Mytilus galloprovincialis blood cells (smear? in vivo or fixed -stained too with MMG - in a counting chamber), if you're able to provide such.

Best regards,

Wolfgang

Dear Gionata,

with pleasure, i'll send you some slides next week,

Thank you for your interest,

Younes,

Dear Wolfgang,

First of all thank you very much for your interest, also for your reply,

Second, about the cells, the slides contains a monolayer native (natural cells), withdrawl from mussels and directly deposited on to the slides,

1. after a 20 minutes adhesion time, the surnatant ejected away.

2. Fixing the smear with methanol by immersing in pure May-Grünwald solution for 3 minutes.

3. diving the slide in a diluted with neutral water (1/2) May Grunwald solution for one minute.

4. diving the slide on to a diluted (1/10) Giemsa solution for 20 minutes.

5. washing with neutral water and aftr drying look in microscope.

N.B.: Neutral water prepared by mixing deionized water (acid) with some drops of (basic) tap water.

Also just to tell something else, in parallel i did a neutral red retention time assay with other aliquot of the same cells, since i'm very familiarized with this assay, i can say that the cells look very normal, but the retention time vary because i did a different exposure experiments.

About the cells in counting chamber, i'll send you the photos as soon as possible (in the early next week).

One more time thak you for your reply and your interest,

BOUALLEGUI Younes,

Dear Younes,

thank you so much for the additional data and your intention to post some other photographs from the counting chamber.

Best wishes and regards,

Wolfgang

PS: I should be glad to see some comment by Prof. Gionata De Vico when she has seen your cells...(:-)) Tante grazie!

Dear all (especially Pr. De vico and Pr. Wolfgang)

i attached some additional photo, different in vivo exposure to different contaminant within the presence of some inhibitors, prior hemolymph withdrawal from animal for different period of time. N.B.: Photo 1&2 for control exposure (untreated animals).

Dear YOUNES, dear  all, many thanks. Waiting to see the slides it seems to me that some of the cells resemble the ones in the image you find attached.  I added a modified version of your first photo with some cells circled resembling "A cells" in my image.  However the image is a photo from histology and yours are from cytology. this is not secondary. Best regards.

Dear Gionata, thank you very much for your interest,

With all my best regards!

Younes,

Dear dr. Boualleguy,

I tried to send a reply before, but probably the message failed starting. If you already received it, excuse me for sending it again.

Your cells are very similar to the sea urchin coelomocytes, when I tried to stain them with giemsa. But it was not possible to identify the different cel ltypes which form the population. So I tried with fluorescent WGA, which marks the digestive vesicles of at least phagocytes. Another attempt may be the elective stain of nuclei, by use for instance of DAPI, or propidium iodide, in order to check that state, shape and size of the relationships between nucleus and cytoplasm. I send you two papers hoping that these can be useful.

Otherwise, contact prof. Canesi, that is a marine toxicologist, specifically working with mussel haemocytes ([email protected]).

Dear Carla,

Thank you very much for your reply, otherwise i know Pr. Canesi very well as i worked in her laboratory for more than three months, but i don't think that she could give hand since she's not familiarized with this kind of work (i mean MGG staining) : since she's not immunologist, anyway thank you very much, also many thanks to every one whom tried to give hand, with all my best regards, Younes!

Dear Younes,

Most haemocytes in your figure are granulocytes (arrows in the Fig. that I am attaching). In those granulocytes the central area appears filled with acidophilic granules, which hide the nucleus, and the peripheral area is hyaline. Although you do not mention the procedure, I guess you have deposited a drop of haemolymph onto the slide and allowed the haemocytes to adhere spontaneously onto the slide for a while; by this way, granulocytes spread on the slide surface and adhere onto the slide, thus resulting in this appearance with a peripheral hyaline area (ectosplasm) and a central area (endoplasm) filled with granules. There are also some smaller haemocytes (double arrow in the Fig. I am attaching), which do not show almost any cytoplasm, with a patent nucleus; this smaller haemocytes could be hyalinocytes or undifferentiated stem-like cells. With the procedure of spontaneous adhesion onto the slide, hyalinocytes and stem cells are mostly lost because they have much lower ability to spread and adhere than granulocytes; this is because those smaller haemocytes show reduced or almost null cytoplasm.

If you want to keep all the cell types in the slide you could either cytocentrifuge haemolymph or to use slides covered with an adhesive film; slides covered with poly-L-Lysine are frequently used. You can contact me for further explanation or any other information on this issue. I am also attaching a paper on mussel haemocyte morphology. Please, confirm that you have read this message. Best wishes,

Antonio

Dear Pr. Antonio,

Thank you very much for your reply

i'll be gratefull,

With all the best wishes,

Younes,

@ Dear Antonio:  thank you really for posting the interesting article from CARBALLAL et al,1997, which also (just for anybodies' convenience) can be found in RG at:

http://www.researchgate.net/publication/250221003_Hemolymph_cell_types_of_the_mussel_Mytilus_galloprovincialis.

In that latter article I found it useful to comparatively see also the cellular properties as demonstrated by TEM.

@Dear Younes - unable to be of help with a further characterization of YOUR LM-hemocyte images from Myt. galloprov. (as posted) - I nevertheless would like to point you to another article(s) which might be interesting to you concerning technique of hemolymph properties' characterization:

by V. Matozzo and M.G. Marin, Eur J Histochem. 2010 Mar 20; 54(1): e9. (open access) @ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167293/

or:  the manuscript version of:      by  Prado-Alvarez, M., Romero, A., Balseiro, P., Dios S., Novoa, B. and Figueras, A. (2012) @ http://digital.csic.es/bitstream/10261/46478/1/Digital%20CSIC.pdf

and, last but not least (out of some more articles i could / would be able to cite here): 

 by  Akihiro Tame et al. in: Fish & Shellfish Immunology, Volume 45, Issue 1, July 2015, Pages 146–156 @ http://www.sciencedirect.com/science/article/pii/S1050464815001199,

the last article containing comparative discussion also to Mytilus galloprovincialis.

Hopefully you will overcome your problems of cellular alterations  (as  Antonio stated), all my best wishes and good luck,

Wolfgang

Article Hemolympe cell types of mussel Mytilus galloprovincialis

Dear all,

Dear Pr. Wolfgang,

Really i'm very pleased for your reply neverthless your interests, i would like to express my best wishes for your interest and your pertinent remarks,

With all my best regards, also with my best wishes,

Younes,

Dear Younes,

you are welcome.

Just to explain my interest in this one:  perhaps you have seen that I one day had been studying "zoology" and ....And at that time not only favorites regarding mediterrean meals when in Italy or Istria...

Have a good and successful rest of the week,

Wolfgang

(you need not addressing me , because I am NOT)