When I was growing Caco-2 BBe cells in an ICC staining chamber, the cells tended to overlap with each other, quite different from the status as they grow in p100 plates. As the cells are not nicely spread out and they tend to form multilayer, immunofluorescent staining showed a much higher background. Does anybody know how to address this problem?
Caco-2 cells don't differentiate well on impermeable substrates such as glass or plastic since they need for free exchange of fluid and ions across the basolateral membrane and this requires a permeable support such as filters. In addition clumping is usually due to seeding cells that are not completely dissociated (often due to passaging at too high density). Alternatively, multilayer formation is more commonly encountered in plastic grown than in filter grown Caco-2 cells. As far as the best filter substrate for immunofluorescent microscopy I would recommend transparent/clear PET filters commercialized by BD-Falcon or Corning clear Transwells. Both can be cut our of the insert and treated for immunofluorescence staining. They should be mounted on glass slides cell-side up and covered with mounting medium and a coverslip. Correct maintenance of the cell line has been shown to affect reproducibility and good differentiation of Caco-2 cells. For information see:
Natoli et al. (2012) Good Caco-2 cell culture practices. Toxicology in Vitro 03/2012
Natoli et al.,(2012) Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. Journal of Cellular Physiology10/2010; 226(6):1531-43.
Protocol for immunofluorescence on clear filters: Ferruzza et al. (2002) Toxicology in Vitro 16: 399–404.
All these papers can be downloaded from ResearchGate.
With HepG2 cells we encoutered similar problems for (high content screening) microscopy. We solved the problem in using collagenase or poly-L-lysine coated well plates. Maybe this works with Caco cells, too.
You can do the ICC in 24 well plates with a cover slip in it. We process and stain them in the plate itself. Following the staining, you can transfer the coverslip on the slides along with the mounting media. This may prevent the formation of the multilayer. Hope this helps
Change medium 4-6 hours after seeding your Caco2 cells, that will reduce the multilayer formation. Also make sure that when you seed the cells they are very well dispensed, avoid cell aggregates, which can be hard with Caco2. One solution is to let the big aggregates sink to the bottom of your tube before you only seed the single cells in your solution. This also reduce the multilayer formation and make the ter value more correct.
Collagen I and poly-D-lysine plates were from BD Biosciences.
Hi Hannes,
Thank you very much for the info. Your suggestions are quite helpful. I would appreciate it if you can send the paper to my email: [email protected]. Thank you! Well, there is another thing: I have been wondering how to stain the cells on Transwells, for the inserts are opaque and there is no way for me to see the cells on the insert... Would you share your protocols of staining the cells growing in Transwells and coating inserts with Matrigel? Thank you very much!
Caco-2 cells don't differentiate well on impermeable substrates such as glass or plastic since they need for free exchange of fluid and ions across the basolateral membrane and this requires a permeable support such as filters. In addition clumping is usually due to seeding cells that are not completely dissociated (often due to passaging at too high density). Alternatively, multilayer formation is more commonly encountered in plastic grown than in filter grown Caco-2 cells. As far as the best filter substrate for immunofluorescent microscopy I would recommend transparent/clear PET filters commercialized by BD-Falcon or Corning clear Transwells. Both can be cut our of the insert and treated for immunofluorescence staining. They should be mounted on glass slides cell-side up and covered with mounting medium and a coverslip. Correct maintenance of the cell line has been shown to affect reproducibility and good differentiation of Caco-2 cells. For information see:
Natoli et al. (2012) Good Caco-2 cell culture practices. Toxicology in Vitro 03/2012
Natoli et al.,(2012) Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. Journal of Cellular Physiology10/2010; 226(6):1531-43.
Protocol for immunofluorescence on clear filters: Ferruzza et al. (2002) Toxicology in Vitro 16: 399–404.
All these papers can be downloaded from ResearchGate.
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