Hi! We are generating hydrogel beads(50-60um) using microfluidics as single-cell beads. To simply all the experiments (especially the centrifuge), I am looking for a dye. The dye should be stable in the beads in 0.1% wash, and do not interfere the DNA assays (isothermal amplification, reverse transcription). Which dye is suitable?
Thanks!
Dear Zonghan!
You look at this publication, please:
Article Fabricating polyacrylamide microbeads by inverse emulsificat...
polyacrylamide (PAAm) microbeads
Fig. 4
Cells recognize and attach to protein-coated microbeads. Fluorescence and phase contrast images illustrate the self-assembly of MG-63, osteoscarcoma cells stained with calcein AM blue (blue, 10 µg/mL, AnaSpec Inc. Fremont, CA) in the presence of collagen-coated (green, pyrene-based dye, Sharpie) and uncoated (red, rhodamine-based dye, Sharpie) microbeads 1 hour (top) and 20 hours (bottom) after seeding. Spheroids containing cells alone (left column) assembled into compact structures over 20 hours. Spheroids containing cells and only coated microbeads (center-left column), only uncoated microbeads (center-right column), or both types of microbeads (right column) formed similar, compact spheroids but with different incorporation behavior of the microbeads.
Thank you! Ra.Jabbar Sh
Alexandr Chernov I am looking for actually a non-fluorescent dye and stable in the bead, even after tween wash
Are you able to include a little phenol red before polymerization?
Wolfgang Schechinger Thanks! Phenol red is a good dye. The problem is that mostly the system's pH
Wolfgang Schechinger How about toluylene red and methyl orange? can they be covalently bond to the polyacrylamide matrix?
Zonghan Gan , I sometime use Phenol Red to dye gels for SDS PAGE (to make the gel borders better visible and to spot air bubbles easier when preparing the sandwiches for electrotransfer afterwards) and add a few drops of a saturated solution in water per 50ml. Gels then are slightly pink after polymerization and turn yellowish when the pH gets too acidic (indicates "overblotting"). Phenolphtalein in contrast will turn colourless when lowering pH.
I've never tried the other dyes you have mentioned. Bromophenol Blue is not incorporated in my experience.
That sounds nice. Is the phenol red covalently bond or non-covalently absorbed into the polyacrylamide matrix? Wolfgang Schechinger
Supposedly some is covalently bound, as a little is moving during electrophoresis and some doesn't.
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