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There are some cell dyes available for stianing cells intracellularly / membrane. I am aware of other channels (CFSE \ PKH26). But I want to stain cells for flow cytometry with a 'red' dye. I have looked at a few options from Thermo but did not find sufficient data on either.

Specifically I am planning to stain a single cell population with two different dyes, treat each stained population somewhat different in between, and then culture them together and distinguish read-outs later. Antibody-staining for a surface marker would be possible with Fab fragments (the antibodies itself may interfere with cell activation during culturing). Therefore I would rather use a red-spectrum dye like CFSE or PKH26 as this may be cheaper and possible for secondary use with other cell lines.

I have multiple cytometers available and the specific emittance doesn't matter as we can pick what is generally possible (LSR/Fortessa).

Thus, I am looking for a dye with emittance in the red fluorescence spectrum which has staining protocols available that can be adapted for my purposes.

Is anybody willing to share their experiences?

Edit

  • Clarified that I needed a 'no-antibody-staining'
  • Clarified question with regard to biological question and FACS
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    $\begingroup$ What is your question? Are you looking for a product recommendation? $\endgroup$ – canadianer Apr 14 '17 at 18:33
  • $\begingroup$ So you're doing a dye dilution proliferation experiment? Please explain what you're doing! Especially what cytometer you plan to use in case the stock filter set up is ill-suited to certain spectra. $\endgroup$ – CKM Apr 15 '17 at 6:08
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I've used the ThermoFisher brand of CellTrace dyes. Specifically, I co-cultured two populations of cells, one with CellTrace Far Red in the APC channel and another with CellTrace Yellow in the PE channel. My choice was in part guided by a product review by another lab touting the relatively low cytotoxicity of these products.

The protocol is quite straight forward. It's also available in CFSE and Violet, though I haven't tested those. These dyes are also optimized for lymphocytes, and so I'd recommend titrating if you're staining anything else.

The dyes are quite bright, and so there were issues with costaining APC-Cy7, for example. I ended up leaving my CellTrace dyes as the only markers on their respective lasers and stacking my phenotyping on the 355nm and 405nm lasers.

For compensation, you might just stick with cells, but in the product manual for CellTrace, however, it says:

"The CellTrace™ reagents readily diffuse into cells and bind covalently to intracellular amines, resulting in stable, well-retained fluorescent staining that can be fixed with aldehyde fixatives."

So if you use the ArC amine-reactive comp beads for LIVE/DEAD kits, you might be able to just make comp beads with those. I'll verify this when I'm back at work if need be, as I haven't done it myself, but it's a nice thought!

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There is a number of different dyes possible, as you can see in this figure (from here):

enter image description here

A good overview can be found in the "Selecting Reagents for Multicolor Flow Cytometry" application note publication from BD (in reference 1) and also the Fluorochrome Specifications from BD (reference 2).

References:

  1. Selecting Reagents for Multicolor Flow Cytometry
  2. BD Life Sciences Fluorochrome Specifications
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  • $\begingroup$ Thanks for your answer, Chris. I realize that I was a bit vague about my question, and have edited it above. $\endgroup$ – David Apr 15 '17 at 6:25

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