I want to do Live/dead cell viability assay on my collagen gels using calcein am and ethd-1
My questions are:
a) What kind of plate for confocal microscopy should I use? I used thin glass bottom 35 mm and ibidi dishes with small well in the middle part but my collagen doesn't attach! should I change the plate or coat the plate?
b) How many cells/cm2 do you suggest? different papers use different cell numbers!
Does anyone do the same experiment or have a helpful source in mind?
Thank you
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Perhaps the above may be helpful. Alternatively, you could explore a generic search engine (google) or Pubmed. Good luck.
Hi Mina Vaez,
You can use a well plate also if you confocal have adaptors for it. Plates might work only at lower magnifications like 20x or less depending on the confocal model. You can coat collagen gel on plates.
The cell number for initial seeding will depend on the duration of your experiment. 3D culture of cells on gel requires more cells than that of 2D. Better to do a trial experiment to evaluate the number of cells required which may depend on the gel thickness too.
This question was discussed in 2015 on ResearchGate at the following link:
https://www.researchgate.net/post/How_can_I_conduct_Live_Dead_Assay_in_cell_seeded_hydrogel
Hi Mina,
appologies, this answer might come a bit late, but our tech support team also has some thoughts on your questions:
The ibidi Polymer Coverslip and the ibidi Glass Coverslip provide ideal optical conditions for fluorescence microscopy. Therefore, confocal microscopy is possible without restrictions when using any of the ibidi labware that contains the ibidi Polymer Coverslip or the ibidi Glass Coverslip Bottom. A suggestion would be to pre-coat before adding the gel - we do so for the µ-Slide I Luer 3D. This is done to enhance the attachment of the gel to the surface of the slide. A suggestion could be to use a collagen coating, that might bind nicely with the collagen fibers in the gel. An example of this can be found in the instructions of our µ-Slide I Luer 3D instruction page 2: https://ibidi.com/img/cms/products/labware/channel_slides/S_871XX_Slide_ILuer_3D/IN_8717X_Luer_I_3D.pdf
An alternative would be to try using one of our µ-Slides with Polymer Bottom instead of class. The coating and the gel would probably work better on the polymer coverslip, and as mentioned before - the optics for confocal microscopy are the same as on glass.
You could also switch to a slide format with smaller wells (e.g. the µ-Slide Angiogenesis or µ-Slide I Luer 3D). The wells in these formats could create a stable environment for the gel and have volume-dependent savings, and maybe even more wells pro slide/dish.
Regarding the cell number it is difficult to give a generalized recommendation. The cell concentration is very much dependent on the cells, the total volume, the format used for seeding and the assay. We always recommend setting up specific assay criteria based on the literature and determine a seeding density based on that. We recommend ordering a free sample of the µ-Slide Angiogenesis and test 5 different concentrations in triplicates. Please remember that the cell suspension should have quite a high cell concentration since it is diluted in the gel e.g. in our Collagen I protocol it is diluted by a factor of six.
I hope this helps.
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