Hi, 

Since Live/Dead stains usually rely on cells processing the dyes, they do not work on fixed cells. 

You can follow the same protocol for encapsulated cells as you would for a cell monolayer. I would just recommend to increase the incubation time with the dyes. Some hydrogels do bind dyes in unspecific hydrophobic interactions. You might therefore get a background fluorescence but it should be much weaker than the signal from the cells. 

You also have to be careful with washing so that the gel does not get damaged. 

Best, 

Alexandra

Thanks Alexandra. Your answer helps a lot. I will try that. 

Let me know if you run into any problems. 

I usually work with Calcein-AM/Ethidium Homodimer, which works well for my mammalian cells. 

Depending on how scattering your hydrogel is, you can run into problems imaging cells deep inside the gel.

I am using 3t3 and suspension cells (K562). I am using PEGDA hydrogel with approx height of 250 µm. I do see some background in my gels. Whats the best concentration of CAM/EtHD that you used for hydrogels? 

Thanks again for the help Alexandra. 

I have used 3T3-L1 before with the suggested concentrations from the supplier (2 µM Calvin-AM, 4 µM EtHD, 40-50 min incubation at RT) in collagen hydrogels. 

If you have tons of background and your cell signal is fine, you could lower the concentrations and incubate a little longer. If the gel is stiff enough, you can also wash more often. 

Otherwise you could threshold the background out from the images in an image processing software, like ImageJ (Fiji), afterwards. It will also help to play a little with image acquisition settings, but most dyes are hydrophobic and get stuck to your gel no matter what you do. 

 Oh, I thought if it is hydrophobic, it wont attach to my hydrogel as hydrogel is mostly made of water. 

But I incubate it for only 15 minutes. I guess that will not be enough. I will incubate for longer time. And yes, my hydrogels are stiff and I guess I have wash more. Thanks for the tip Alexandra. You are helping a lot in my process. 

HI Alexandra, 

My cells are fluoresces for both green and red. So I really can't say whether cells are live or dead. Did you ever face this problem?

Thanks, 

Krish

Hi, 

If they stain for both, the cell membrane might be compromised while they are still alive. This can either be due to stress during encapsulation or they are actually starting to die. 

How long after encapsulation did you do the staining? Did you try to do different timepoints? You could also try to assess if cells are proliferating or look at their general morphology. 

I have had problems with cells dying in hydrogels due to too stiff matrices or to high protein content of the matrix (and that was only 5wt% protein in total but it was already too much for my cells). You can also stress your cells with too high/too low density of bioactive sites. You could try different concentrations of PEGDA in your hydrogels. 

Best, 

Alexandra

P.S. sorry I did not get a notification about your question and did only see it now.