I m using collagen impregnated with plant extracts for my studies. I want to check biocompatibility of the material using MTT assay. Can anyone suggest me a detailed protocol for the same?
I would consider using coated glass coverslips for this. Small (11mm diameter) round slips will sit nicely into a 24 well plate, 2cm x 2cm squares will require a 6 well plate and larger volumes of reagents. Alternatively you could coat the wells themselves, but then perhaps you might consider low attachment plates to avoid the tissue culture coating of normal plates from masking the effects of your material. These can however, in my experience, be very variable from batch to batch and brand to brand!
I recommend using a concentration and volume of collagen solution equivalent to 6-10 ug/cm2. Cover the glass with a small volume (around 200ul) held under surface tension (so covering the slide, but not flowing into the base of the well) and allow to air-dry in a flow hood overnight.
The following day seed your cells at the required concentration. I'd start around the 5000-10000 cells/well mark, but it depends entirely on the size and metabolic activity of your cells of interest (I'd consider performing an optimisation step here). I'd then collect representative images once the cells have adhered (or failed to?) and assess their viability using MTT assay as you intend.
If you are interested in multiple time-points, using MTT you'll have to set up replicate plates. Instead for this purpose I'd advise you to look at the alamar blue assay. It is similar to MTT in that it measures cell number via mitochondrial reductase activity (in this case the conversion of blue resazurin to pink resorufin) but it can be rinsed off, replaced with growth medium and the culture continued for repeated measurements over a period of time. It can be read by fluorescence (ex 545nm em 590nm) as well as absorbance, so can also be more sensitive than MTT.
I hope this is helpful?
Merlin
Hi,
Do you have the collagen sheet? If yes, you can basically put the sheet in the culture medium and collect the medium in time. Then, you can treat the cells with that mediums and measure by MTT. I think it will work.
Good luck.
Mojtaba
Dear Viji, as for MTT you would have to dissolve the cells (usually with dmso) you must be sure that the collagen sheet (that would probably dissolve as well) won't modify your analysis. I would do a blank lecture: use MTT on collagen sheet without cells and see, after DMSO treatment , the plate reader output.
As said before i would consider Alamar as an alternative. Remember to built a calibration curve in order to monitor cell number.
You also might want to try loading the cells on your scaffold (if you have a sheet) in universals first by, then after the cells attach you can transfer the scaffold to wells. This can avoid the cells preferentially adhering to the bottom of your TCP wells instead of your scaffold and therefore getting inaccurate results.
1) Place your collagen membrane scaffold into a universal (separate ones for each).
2) Suspend your cells in the required amount of media to submerge the scaffold.
3) Place the cell suspension media into the universal with the scaffold.
http://www.ncbi.nlm.nih.gov/pubmed/23261927
You can take some ideas from the linked article that i send you...they also work with collagen patches and cells, they dont use MTT but they do quantify the number of cells attached to the patch
Hope would help
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