I have two question
First, how should I sterilize my hydrogel or hydrogel's ingredients for a live/dead assay? The ingredients are not autoclavable.
Second, how can I mix cell suspension with my hydrogel for live/dead assy?
hi,
1. Make arrangements and try to prepare your hydrogel in a sterile condition (Laminar hood) to minimize contamination. if not after preparing hydrogel, give UV sterilization inside the laminar hood and then seed the cells. Add a little extra antibiotic to the culture media used to grow cells in the hydrogel.
2. Try to use a stain that does not need fixing of cells. Calcium-AM and ethidium homodimer staining doesn't need fixing. Even you think of alternative assays such as glucose assay, MTS assay, and Alamar blue assay to understand cell viability.
In addition, you can dissolve the hydrogel using a suitable enzyme and retrieve the cells to analyze the viability.
Regards
Saurabh
UV light for 20 mint after gelation, should be good enough. However, before gelation, you can just filter it. F
You have some excellent suggestions here provided by Saurabh Mandal and Ahmed Nada . Live/Dead kits from thermofisher have worked for my hydrogels before and you can check this reference, Article Anisotropic Fiber-Reinforced Glycosaminoglycan Hydrogels for...
I have had better success imaging these on a confocal microscope as it reduced the background by a lot. If you have access to a multiphoton imaging system, you can make use of cell's auto-fluorescence to your advantage and here is a reference that talks more about this, Article Assessment of Cell Viability in Three-Dimensional Scaffolds ...
It would be helpful if you can share what your hydrogels are made of. For instance, if you are working with photocrosslinkable hydrogels, I would recommend syringe filtration. If your solution is too viscous, try heating the solution before using a syringe filter (first check to see if temperature affects your prepolymer solution).
Here is a reference that talks about gelma synthesis and how to maintain sterility, Article Functionalization, preparation and use of cell-laden gelatin...
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