Did you use Poly-L-Lysine Coated Microscope Slides ?

Yes, sorry, should have mentioned that in my post.

How are you fixing the cells? Perhaps using a cross-linking fixative like glutaraldehyde might improve the cells' robustness.

As far as sticking them to the slides, you might try coating the slides with a lectin that recognizes something on the surface.

We use a two-step fixative procedure, which includes gluteraldehyde. (primary fix = 4%paraformaldehyde + .000025% gluteraldehyde in culture medium) (secondary fix = 1% formaldedyde + .000025% gluteraldehyde + .25% triton-x in a buffer solution).

We spin the cells onto poly-l-lysine or electrostatic (plus) pre-coated slides, to which only one of five subset of cells binds. We haven't developed cell surface recognition markers that are common throughout all cell types (5 different cell types, different classes of coelomocytes). We don't have any lectins which interact with all of the cell types; do you think it would work to make a mixture of, say, 3 lectins and coat slides with the mixture?

Another thought: any clue if its possible to coat the slides in an antibody that will recognize the cell surface of all the proteins? (I have to look into whether or not we have found a surface protein shared by all cell types, and if so, if we have an antibody against it)

Thanks!

below link provides the information about EDU labelin

http://tools.invitrogen.com/content/sfs/manuals/mp10044.pdf

@Vakamullu, I have read the manual, which lists flow cytometry (which we do not have access to at the moment) and fluorescent imaging as the methods for EdU detection. We use the latter kit, but as we cannot adequately get our cells onto slides (only 1 of 5 populations), we are looking for other possible alternatives

To elaborate a bit on the lack of cell adherence, the cell population that adheres to the poly-l-lysine slides is a phagocyte type cell. The other cells do not adhere to the slides, and we get a number of nuclei to stick to the slides (although we don't know if these are from busted up phagocytes or other cell types).

Did you try to plate the cells before fixation and permeabilization ? we have worked with human leukemic K562 cells in order to stain the membrane ABCG2 transporter for fluorescence microscopy analysis. Cells were first plated on a poly-L-lysine-coated coverglass (Sigma, St Louis, USA). After fixation with 4% (w/v) paraformaldehyde for 15 min and permeabilization in 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min, the staining was performed. The cells were not damaged and we did not observe isolated nuclei).

@Hamid, what do you by plate the cells? As in for cell culture, but onto the poly-l-lysine slides? That's an interesting idea. Right now we spin the cells at 2000 rpm for 5 min at 4*C to get them onto the slides.

Lauren, when you say that most of the cells are destroyed, do you mean they lyse, or they don't stick to your slide, or both? I've seen protocols using 3-4% formaldehyde and 0.02% glutaraldehyde, so I think you can increase the glutaraldehyde safely.

I think that a mixture of lectins and/or antibodies should work if you know enough about the surface molecules of your cells. If you really wanted to try that, you could raise polyclonal antibodies against the fixed cells themselves.

Another thing I've heard of is fixing the cells on polylysine or other amine-modified slides. The cells will settle onto the surface, and the aldehyde reagents will tend to crosslink the cells to the amino groups there, holding them in place.

PS: I see Hamid has suggested something similar. I forgot to mention that the protocol I saw suggested letting the cells settle onto the slide and *then* adding a concentrated solution of fixative, so that the cells aren't washed before they're fixed.

Yes, the cells were plated onto the poly-l-lysine slides by performing a cytospin. Hamid

Hi Louren.

Do you need something like that (see attachment)?

This is EDU staining of haemocytes (another marine invertebrate) counterstained with EtBr and phase-contrast optics applied. These cells were stained in suspension (several centrifuge-washing iterations) after fixation with PFA (4%).

You need collect your coelomocytes directly into fixative and to process the sample in a centrifuge tube as it described in the trivial EDU protocol, but replacing any slide washing with centrifuge passage.

Have a fun... and forget about coelomocyte attachment to a glass slide. Nobody in the Universe can do it adequately for quantitative analysis . )))

Have you considered a plate reader based assay? If you have access to a fluorimeter you may be able to quantitate the relative amount of EDU incorporated based upon signal intensity from a defined number of cells. I have not done this particular assay, but have done some similar assays. If you have access to some adherent cells you could trypsinize and use as a control, you could probably get fairly quantitative.

As Laura mentioned, there are other assays that do not rely on adherence to a glas slide. Assays such as MTT which uses fluorimeter has proven very usefull for our studies of suspension cell proliferation.

@Wes, I mean that only some of the cells (pretty much 1 cell population) sticks to the slide, and within that one population, some number of cells lyse in the process. I'll consider increasing the glutaraldehyde, thanks.

@Laura, our plate reader is not accurate enough for the variability (we tried this)

@Alexander, that sounds like a great alternative, thanks!

Hi Lauren,

Below reference may also useful for you.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737075/

The Cytospin worked well for me! https://www.google.com/search?q=cytospin&hl=en&client=firefox-a&hs=DTb&tbo=u&rls=org.mozilla:es-ES:official&channel=fflb&tbm=isch&source=univ&sa=X&ei=RzuiUJvhCeaN4gSNnIGADA&ved=0CDcQsAQ&biw=1280&bih=827 you must centriguge the cells on poly-lys coated porta slides

I am having similar problem. On cytospinning, the leukemic cells are lysing. HeLa cells on the other hand don't lyse on cytospinning. I will consider increasing fixative concentration but any other suggestions are most welcome.