Hi!,
I am currently working with integrins. I wonder that if there any differences in terms of the staining pattern of integrins, when you use no ECM coated coverslips and fibronectin-coated coverslips.
Thanks
You will observe completely different staining patterns, depending upon the antibody you use and what method you use to persuade your cells (whatever these might be) to attach to a non-coated surface. Different cell types express and use different integrins, but all cells/integrins do require a ligand to act as the recognition and attachment target.
No coating > no ligand > no attachment > no integrin organisation!
What are your coverslips made form? Glass? Polystyrene? Thermanox? Charged/Non-charged?
What are you trying to demonstrate / observe?
Can't be more specific because you provide too little information.
David Ian Leavesley , thanks for your kind answer.
I was thinking exactly the same way which you wrote about no coating-no organization. I use 16mm glass coverslip without any coating due to RPE1 cells are easily attaching the surface. I am investigating integrin and my protein of interest interaction in this cell line. For this purpose IF is the most used method for me. However, there was little colocalization between integrin and my POI which must be there. I thought maybe an ECM can help me to the chance distribution/activation of integrin .
Hi Baran,
This entirely depends on which cells you are using, and which integrins you are looking at.
It is important to keep in mind that each specific integrin heterodimer has its own specific ligand. Some integrins bind collagen, some a specific laminin, some RGD sequences or other specific proteins. So what integrin are you staining for?
Secondly, some cells can attach to plastics or glass, some need a coating. But keep in mind that the coating or surface affects cellular attachment and ultimately its behavior. Epithelial cells, probably including the RPE1 cell you are using, can produce multiple types of adhesion molecules, including multiple types of integrin heterodimers. Whether these molecules are used for adhesion is dependent on their surroundings, or in this case, coating. Also be aware that some cell types, including epithelial cells, can produce their own ECM molecules and adhere to these.
Perhaps you can supply information on which integrin you are specifically looking at. And for trouble shooting, it can be a good idea to try multiple coatings anyway.
Good luck!
Hi Eelke Béguin ,
I am looking at beta 1 and alpha 3 integrin subunits.
Hi Baran,
As a3b1 is a laminin-binding integrin, it may be a good plan to leave your cells for a couple of days once you have seeded them, before you fix and stain. This allows them to produce the laminin containing ECM. You should be aware that integrin b1 is present in many integrin pairs, and might therefore not give the particular staining/co-localization you are expecting.
It depends entirely on the cell types you are using, and how you plate them.
Generally, if you are using serum-containing media, vitronectin will promptly coat glass cover slips, so if the cells express avb5 integrin, as many do, that may enter focal contacts. If you pre-coat with fibronectin, then a5b1, a4b1, avb3 may be found in adhesion contacts, of appropriate cells. But unless you take care to saturate all other protein-binding sites on the glass surface, you will get a muddle, because vitronectin will promptly coat those, plus any ecm molecules the cells may make, so you get a mixture on integrins in the contacts.
Depending on the cells, they may make a wide variety of ECM molecules, which can with varying kinetics, deposit onto uncoated surfaces in ways dependent on cell type, substrate (glass? plastic?) and culture conditions.
With time, (hours to days) these molecules can also cover any coating you might initially use. You can block biosynthesis of these endogenous proteins (which is drastic) or do time courses.
Finally, most/many cells express or secrete proteases which can modify proteins on the substrates. So you need to be very cautious when interpreting your results unless you take such factors into account.
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