Hi Sunil,

what about this one? See links attached.

Marcus

www.sigmaaldrich.com/catalog/product/sigma/c4361

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/3/c4361dat.pdf

Alternatively this one...

http://www.anaspec.com/products/product.asp?id=28613

 Hi Marcus,

Thank you for the information. But those FITC conjugated collagen are used for collagenase activity. However, I want to prelabel the collagen solution before forming gel and want to see how good has the gel formed. 

Hi Sunil,

If you want to pre-label collagen, you can use NHS-Fluorescein (see first link attached). The second link provides the protocol how collaborators label reconstituted collagen gels as example for TAMRA succinimidyl ester in PBS, but you may use the same protocol, it should work. You may use a lower concentration, you'll need to try out a bit.

Keep in mind that NHS esters slowly hydrolyze in aqueous solution, so you have to be quick after dissolving it in buffer for staining procedure (otherwise, the NHS ester does not react with the amino groups of collagen, but only with water). The best is to prepare high concentratet aliquots in water-free DMSO which are used right before staining and stored at -20 °C.

Marcus

https://www.thermofisher.com/order/catalog/product/46410

Article The phenotype of cancer cell invasion controlled by fibril d...

Thank tons Marcus. I'm gonna try this one

 Hi Marcus, I apologize if I did not make myself clear about the question. I actually want to seed endogenously red fluorescence Human Mammary Fibroblast (HMF) to the collagen solution ( which would be pre-labeled with a dye) and once they are gelled together , I want to see the fluorescence of both the component under microscope.

I do not think NHS fluorescein will help me dye the collagen solution? to which I will add the fibroblasts and let them gel.

Thanks,

Sunil

If you add the NHS ester to collagen prior you add the fibroblasts and let react for 10-15 min, this should be no problem since the reaction ist quantitative (everything you put in will react, since collagen is in a great excess) and side products should not harm the cells. Additionally, you need to add only a little amount to get a good fluorescence signal.

In any case you need to run a control experiment w/o the addition of flourescein NHS and do Life/Dead staining to check if the cells are not harmed.

The most safe way would be to dialyze (use a dialysis tubing with MWCO 1000 Da and dialyze against 0.02 M acetic acid or 0.01 M HCl) and freeze-dry the collagen after labeling. Then it will be really clean. The experiment I mentioned before is more the "quick and dirty" version.

I really appreciate your suggestions Marcus. 

Hi sunil,

Did you try it? if yes did it work for you?

Lemme know please if it worked. I need to do a similar type of stuff using collagen.

Thanks

Abdul

Hi sunil

Did you try it? if yes did it work for you?

Lemme know please if it worked. I need to do a similar type of stuff using collagen.

Thanks

Abdul