I would like to fixate cells in culture and then add other cells to see if they interact with proteins on the membrane of the fixate cells. Is there a way of fixating cell, and thus killing them, while retaining all of their membrane components? I need to fixate the cells because I would like them to remain in place during life cell imaging.
We previously used the ghosts of such cells (in our case fibroblasts) as a basal layer. We grew them to confluency and then lysed with hypotonic solution. After a rinse with media, the neurons we were studying were plated on top of the ghosts. This allowed for excellent adherence and migration.
I've never heard of fixed cells as scaffold, but what I've seen is mitomycin-C-arrested cells used as a basal layer. It is pretty common in immunology, when you need to challenge lymphocytes to target cells.
Hope this helps.
@Katharina: I need the first cells to remain immobilized, to see if the other cells migrate along them. If they are still viable, they contract and elongate during the experiment and that makes interpreting the movement of the other cells rather difficult. I don't actually need them to be dead. I just want them to stay still.
Dear Colleagues,
If you search "poisonning" agents of cytosqueleton, to inhibit movments, you have: cytochalasines A and B, colchicine, vincristine, vinblastine, nocodazole ...
Usefull ?
Regards
Didier
We previously used the ghosts of such cells (in our case fibroblasts) as a basal layer. We grew them to confluency and then lysed with hypotonic solution. After a rinse with media, the neurons we were studying were plated on top of the ghosts. This allowed for excellent adherence and migration.
If the matrix cells can be dead, fix cells by using paraformaldehyde (1-4 % for 30 min), which is quite reversible for preserving the aqueous accessibility of macromolecular structures. This is one of the best way to work with membrane compartments and their interactions with using immunolabeling tech. good luck.
Phyllis Pugh suggests an excellent answer. We too have used cell 'ghosts' with great effect.
Polylysin won't inhibit the movement of the cells, it will increase the adherence but the motility will be unaffected.
And finally, I see no reason why fixing wouldn't work - it would be interesting to find out!
Let us know how you get on!
When seeding neurons or microglia on top of a monolayer of paraformadehid-fixed astrocytes, neurons survive better and microglia ramify, suggesting that many cell-cell interactions are preserved
In this paper, the authors fix neurons (axons) and then seed different cells (OPCs) on top. No special technique seems to be needed.
"Axonal Fixation. Before the seeding of OPCs, coverslips containing purified DRG neuronal cultures were washed twice gently in PBS. Neurons were then fixed with 2ml of a 4%paraformaldehyde (PFA) solution for 10 min. After the removal of PFA, neurons were gently rinsed multiple times with PBS and then transferred to fresh wells with MEM containing 10% FBS."
As mentioned above, be aware that fixatives modify proteins, including by fusing/cross-linking the proteins, potentially changing how your other cells will interact with them.
If you are only interested in a specific protein, can you coat culture plastic, or cell-sized beads, with that protein, then seed your cells on top?
Can you obtain patches of membrane from your cells, with desired protein(s), and coat culture plastic with them, rather than the living cells?
I'm not aware of any substrate that will 'glue' your bedlayer of cells in place.
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