I wonder if it is possible to dissociate glandular tissue by an agent like collagenase / accutase etc. and do an flow cytometry / cell sorting right afterwards with those cells. Or do you think the cell markers will be still too disrupted?
Anyone ever tried it or read about it?
What dissociation agent would you recommend for glandular tissue?
What sort of glandular tissue do you plan to use? I have regularly assayed lymph nodes by FACS right after harvest. I did not use enzymes as i was looking for T cells and they went into solution quickly with mechanical dispersal (I used the frosted ends of two glass slides to smush them into solution).
Worthington is generally considered an excellent resource for digestive enzymes. Check out this document, they have a list of tissues at the end with the enzymes you would use.
http://www.worthington-biochem.com/CIT/CIT_Product%20Insert_2012.pdf
It is really difficult to take out all cell proteins from medium in any type of cells specially glandular cells, but look for an enzyme that attach to the cell protein and gate both cell protein besides what you are looking for and you can identify that during the gating
Thank you everybody for the answers! @Taylor: Could you send me a protocol that you are following?That would be very nice and a great help!
@ Felicity: How does it work with those frosted end of glass slides? Do you also happen to have a protocol, that you could send me? Thanks and best wishes!
Depending on how you isolate the cells, you may need to add a nucleic acid dye that is excluded by live cells. You can gate out the dead and obtain clean results. The choice of dye depends on what fluorochromes you are using in your assay.
Mathias, I would like to advise you to search the internet for dissociation protocols. There are many published but no golden standard. Also look in Currrent Protocols in Cytometry. Hope you have it in your library. Frequently collagenase-based enzyme mixtures are used which can include trypsin, dispase, etc. I would also like to advise you to add DNase. You will loose many cells during the dissociation process. The DNA is released and other cells will stick to it. These cell clumps will clog your flow cytometer. Use nylon filters. Ross advised to add a DNA stain. I would also recommend that. First, it will help you to discriminate between "cells," well at least it is a nucleus, and the debris in your sample. These will dramatically improve the discrimination between different cell populations. Secondly, when you are working on cancer, it will allow you to detect DNA aneuploidy and identify tumour subpopulations based on differences in DNA content. Type in my name (corver we) in PubMed and you will find protocols for tumour dissociation. Maybe you will get an idea. Likely you are new to this field. Why don't you become a member of the German Cytometry Society (DGfZ). Members are certainly willing to help you further and you can share your experiences during the yearly meeting (I think it is in Octobre)
Regards,
Willem
Dear Matthias, in case you would like to minimize the debris on your sample I would suggest to test our pluriStrainer. We provide them down to a mesh size of 6µm. This will exclude most of the debris and aggregated cells.
Best wishes
Michael
Hi Matthias, I sometimes use glass slides to disperse tissues. Normal glass slides used for microscopy have a frosted end that is normally used for labeling with a pencil. The surface of this part of the slide is somewhat rough, so we use sterilized glass slides, position the small tissue on the frosted side of the glass slide, and use another sterile glass slide to mash the tissue, thus breaking it down mechanically. Works well for lymph nodes and spleen. For liver, lungs, brain and tumours I use other methods that involve enzymes.
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