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Absorption spectrum of Phenol red posted at
https://www.linkedin.com/posts/georgemcnamara_phenol-red-absorption-spectrum-and-chemical-activity-6844447309233696768-eKYy
**
Springer protocol URL Stadtfeld 2005 in Methods in Molecular Medicine,
Vol. 105: Developmental Hematopoiesis: Methods and Protocols.) ... I
suggest: "garbage in, garbage out." Every fluorescent protein and filter
set and light source: obsolete.
2.3. Phenol Red-Free Medium and Glass Bottom Vessels Improve Image Quality
The second parameter that contributes to image quality is background
fluorescence. To test the influence of phenol red in the culture medium
on background fluorescence, we acquired a series of images in media with
and without this pH indicator. As shown in Fig. 3, phenol red
dramatically increases the background levels, especially when
visualizing GFP and RFP with the Endow GFP and TRITC filters,
respectively. As a
result, the relative signal intensity is decreased, hampering the
detection of weak signals (when visualizing YFP with the Yellow GFP and
JP2 filters, this effect is only minor). Therefore, we recommend using
phenol red-free medium (we routinely fill a chamber in an eight-chamber
slide or a row in a 96-well plate with phenol red-containing medium to
visually monitor the pH of the cultures).
"phenol red dramatically increases the background levels" ... figure
shows under 20% increase in intensity. My favorite item in the book
chapter, the PC: "1024 KB RAM, 80 GB hard-drive)".
*
See AausFP at Lambert et al 2020
(https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000936)
and in addgene (though the addgene plasmids may not be human or mouse
codon etc expression optimized).
***
no need for Phenol red in culture media on microscope, and bicarbonate
ions are not just a buffer: they are a substrate for CFTR and several
other ion transporters.
Evrogen has been selling DMEMgfp for nearly a decade (URL is for DMEMgfp-2)
https://evrogen.com/products/medium_DMEM_gfp/medium_DMEM_gfp.shtml
and ThermoFisher (enjoy using their search engine) and abcam (acquired
Marker Gene Tech)
https://www.abcam.com/opti-kleartrade-live-cell-imaging-buffer-1x-ab275938.html
sell similar media.
Happy 2021 and beyond,
George
On 9/16/2021 9:17 AM, Sylvie Le Guyader wrote:
> *****
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>
> Hi again
>
> I asked Thermo Fisher and got this:
> --------------------------------------
> Phenol red (and other cyclic compounds) has been shown to increase background fluorescence (its peak is 440 nm), which can create issues for users depending on the channels being used. One such reference is https://link.springer.com/protocol/10.1385/1-59259-826-9:395. Hence if using colors in that spectrum, it is best to use phenol red free medium. Additionally phenol red's cyclic nature is similar to that of estrogen, so it can bind to and activate ERs of estrogen sensitive cells (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC323325/). If doing work in those areas it is also a good idea to remove phenol red.
> John Fisk, Ph.D.
> Technical Applications Scientist
> Life Sciences Solutions Group
> Thermo Fisher Scientific
> T (NA) 800-955-6288 | (EMEA) 00-800-5345-5345 [log in to unmask] | thermofisher.com
> -------------------------------------------------
>
> The ref given by John from Thermo Fisher shows a 20% increase in background fluorescence in the green and red channel when using Phenol red compared to medium without. There is also an interesting information about Estrogen which I did not know. Absolutely nothing about quenching fluorescence. However, I saw that the measurements were done on plastic dishes. I did measure background fluorescence in the green and red channels with and without Phenol red some years ago and never found any difference. I wonder if one would get the same results on glass bottom dishes.
>
> Pawley's confocal handbook (p 361) mentions that the problem with phenol red is fluorescence quenching but there is no ref.
>
> I would say that the question is not fully solved...
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7C,
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> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Model, Michael
> Sent: 15 September 2021 18:41
> To: [log in to unmask]
> Subject: Re: EXT: Phenol red in imaging medium
>
> *****
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> *****
>
> It seems to have a strong absorbance at 550 only at alkaline pH (https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fpublication%2F221925346_Plastic_Optical_Fiber_pH_Sensor_Using_a_Sol-Gel_Sensing_Matrix%2Ffigures%3Flo%3D1&data=04%7C01%7Csylvie.le.guyader%40KI.SE%7C6e92ce91403941f654bc08d978684199%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637673211709146746%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DdJTtTEN163wfYxI5naW9kXsatMUWu56nufjAwlsajw%3D&reserved=0). But if left in the room, bicarbonate-based media will turn alkaline, so it is possible that some quenching by energy transfer occurs.
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Sylvie Le Guyader
> Sent: Wednesday, September 15, 2021 10:51 AM
> To: [log in to unmask]
> Subject: EXT: Phenol red in imaging medium
>
> *****
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>
> Dear all
>
> I have heard that the reason why Phenol red is avoided in cell culture media used for imaging is that it quenches fluorescence in some (at least the green) spectrum. I cannot find any reference about this. Does anyone know of a paper showing this?
>
> thanks
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Hälsovägen 7C,
> 14157 Huddinge, Sweden
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