Hi everyone,
I would like to attach MOLM13-cells for fluorescence microscopy. However, they are cultured in suspension and hardly attach to glass, Poly-L-Lysine-coated glass, or the IbiTreat-slides from IBIDI. Has somebody an idea what to do? If you have information about attaching suspension cells in general and not MOLM13 specifically - thats also fine!
Thanks a lot!
Hi
I would suggest you that in 12 well plate prepare Poly-L-Lysine coated glass, put 2 million cells in 1 ml of either medium or PBS, centrifuge 15 mints at 1000 rpm. I had done several times. For sure my cell line isn't MOLM13-cells, good luck!
Hi
I would suggest you that in 12 well plate prepare Poly-L-Lysine coated glass, put 2 million cells in 1 ml of either medium or PBS, centrifuge 15 mints at 1000 rpm. I had done several times. For sure my cell line isn't MOLM13-cells, good luck!
Thank you very much for you answer! Just to clarify - you suggest to centrifuge the cells inside the 12 well plate? This may be difficult since we do no have such a centrifuge...
You are welcome. Yes, centrifuge the cells inside the 12 well plate, you only need an adapter for that.
You could centrifuge cells in a falcon tube and suspend them in the smallest possible volume such that you could coat each cover slip with about 200- 300 ul of this concentrated suspension. Wait 20 minutes to an hour to check for cell attachment (you can check under the microscope, they will look settled compared to when you first plate them) before you can go ahead and flood each well with ~1ml of media.
You can stain them in a falcon tube by gently harvesting the cells at any step and finally use a cytospin for the collection on a glass slide. Cells from cytospin are not as beautiful as the attached cells are but this works well for the purpose
Hi,
I would not suggest using polylysine as it is toxic for many cells types.
You could try to:
- get some antibodies which are specific to some abundant membrane proteins that you are not interested in
https://books.google.nl/books?id=vUTAIOV7WtQC&pg=PA95&lpg=PA95&dq=antibodies+immobilization+cells+glass+microscopy&source=bl&ots=y7gai9JiLS&sig=32w9nCgOaFXhVDHH_pFEv3KKHyE&hl=en&sa=X&ei=CEw6VZzrJ82_PPWwgLAL&ved=0CGcQ6AEwCw#v=onepage&q=antibodies%20immobilization%20cells%20glass%20microscopy&f=false
- make the glass surface positively charged somehow, and then the negative charge induced on the membrane by membrane potential will do the job
https://books.google.nl/books?id=PjeaBQAAQBAJ&pg=PA202&lpg=PA202&dq=cells+immobilization+positive+charge&source=bl&ots=kH7g39nfhW&sig=cpBLQy0qY3_rwoWvLUlEd806e00&hl=en&sa=X&ei=QEw6VZHYF8riO8y0gPAD&ved=0CEYQ6AEwBg#v=onepage&q=cells%20immobilization%20positive%20charge&f=false
- sandwich your cells between a glass slide and an agarose pad, but this could introduce some membrane deformation if the agar is too thick.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504454/
hope this helps,
Best,
Jacopo
I used to attach eukaryotic parasites to Lysine coated coverslips in 24-well plate by centrifugation.
Hello all,
Can't we use PDMS coating over the glass sildes for fluorescence microscopy?
Is it important for cells to attach to the glass for fluorescence to happen? Isn't it possible over PDMS coating, when the medium is PBS?
Hello everyone discussing this topic,
Shi-fix coverslips are an easy option for this purpose. Suspension cells attach quickly on these coverslips ( 30 mins) or can be directly grown on them. The slides work very well for Immunofluorescence.
http://www.shikharbiotech.com/products/shi-fix-coverslips/
Hope that helps you if the issue is still relevant to you.
Regards,
Ravindra
Another alternative is to use the Cellasic ONIX system. There are plates (M04T) that are specifically designed to trap and immobilize cells without needing to synthetically attach them to a surface. The cells can then be fixed and stained in the plate. Might be worthwhile for cells that are challenging to adhere to a surface or in cases where there could be artifacts from such adhesion.
While not exactly immunostaining of cells, here is a Youtube video showing the trapping of non-adherent cells followed by staining and analysis with live cell dyes.
https://youtu.be/aMtgsEwjxYU
Regards,
John
simple sedimentation may work:
Article Protocol for adhesion and immunostaining of lymphocytes and ...
you could also try old fashion smearing techniques.
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-non-adherent-cells
good luck !
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