I have tried to perform Live/dead assay on my seeded decellularised trachea scaffolds approximately 2-3mm thick,, were I have washed my biopsies initially with PBS then stain with Calcein Am/ Ethiduim Homodimer for one hour ,then fix in 4% PFA for few hours and then, 30% sucrose then cryosection , But unfortunately could not get any signal and there was background fluorescence. Can Any one advice on a better way where I can perform this assay. The scaffold is opaque so imaging is a problem??
You could perform a cell metabolism assay using Alamar Blue as a round-about way of determining the relative health of the cells within your scaffold. The Calcein Assay isnt working because it wont fluoresce for the time span you are expecting (plus the cells need to have intact phospholipid bilayers for it to work). For best results, Calcein stained samples should be imaged immediately after staining, and samples shouldnt be cryosectioned/frozen. But, you could try using a razor to cut a crossection for imaging (maybe about 1 mm thick), and then make sure to wash with PBS after calcein Staining of the section to reduce background noise/your scaffolds autofluorescence.
Hi, agree with Marc. To get complementary informations (like eventuelly zonation effect) you may perfom live/dead staining, toroughly wash your samples and perform LSCM acquisition. By using middle magnification, you could image the whole sample and analyze thelive/dead staining on the different z stacks.
Hi, I work with human cartilage. For L/D Staining I cut the UNFIXED explants with a double-bladed scalpel (0.5 mm thickness). After Staining (1 µM calcein AM and 2 µM ethidium homodimer-1 for 30 minutes) I use a z-stack module (software AxioVision, Carl Zeiss) for analysis. Is it possible to cut our scaffolds with a scalpel? My colleague also works with collagen2-scaffolds and she works with my protocol, too.
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