Fixation procedures can allow leakage out of the GFP-tagged protein or be directly detrimental to the fragile amino acid cage that enables GFP's remarkable properties, properties that literally started a cell biology revolution and rightly recognised by a Nobel Prize!
Q. How can we reliably measure GFP expression in cells by flow cytometry or by imaging?
A. Analyse LIVE, intact cells that we've stained with DRAQ5™ or CyTRAK Orange™.
These two cell-permeant DNA binding probes are spectrally compatible with GFP and allow you to counterstain your cells, alive and intact, without any unnecessary processing with fixative or permeabilization. If necessary, you can use a much gentler fixation (e.g. less formaldehyde) and that remains compatible with these probes. (High content screening labs even combine fixative and DRAQ5 to have only one pipetting step during the last steps prior to imaging).
Counterstaining is rapid (15 min.) and no washing is required. Just tamp away any excess liquid from your cells on a slide, apply any required mountant and coverslip prior to imaging.
For flow cytometry and imaging flow cytometry the nuclear staining will be the last step, with no need for washing, proceeding directly to the analyzer.
For flow cytometry and imaging flow cytometry the nuclear staining will be the last step, with no need for washing, proceeding directly to the analyzer.
Which one should you choose? This spectrum might help.
Of course, your equipment options may determine this for you, but for either of these counterstains you won't be waiting to book time on a UV-equipped 'scope or cytometer! To make it easier, here's a quick guide to each of the products for microscopy, flow cytometry and imaging flow cytometry respectively:
Searchlight™is provided courtesy of Semrock/IDEX Health & Science, LLC
Of course, your equipment options may determine this for you, but for either of these counterstains you won't be waiting to book time on a UV-equipped 'scope or cytometer! To make it easier, here's a quick guide to each of the products for microscopy, flow cytometry and imaging flow cytometry respectively:
DRAQ5™ has peak absorbance at 599 and 646 nm so any of the longer wavelength excitation sources will suit for microscopy - 594, 635, 647 nm - alongside 488 nm excitation for your GFP measurement.
DRAQ5™'s emission is detected above 670 nm so your microscope or high content imager needs to have a far-red channel. This is commonly a 675LP (long-pass), often called the "Cy5" channel but it could be any broad bandpass (BP) filter sitting in the 670-750 nm range.
This means that your precious GFP and DRAQ5™ counterstain signals can be captured simultaneously (ideal for HCS) from two independent excitation sources.
Remarkably, in modern flow cytometers DRAQ5™ can be efficiently excited by blue through red lasers and is detected in a channel similar to those described above for microscopy.
On the Imagestream®x (Merck) imaging flow cytometer DRAQ5™ is detected in channel 11 (Camera 2) or channel 5 (Camera 1).
DRAQ5™'s emission is detected above 670 nm so your microscope or high content imager needs to have a far-red channel. This is commonly a 675LP (long-pass), often called the "Cy5" channel but it could be any broad bandpass (BP) filter sitting in the 670-750 nm range.
This means that your precious GFP and DRAQ5™ counterstain signals can be captured simultaneously (ideal for HCS) from two independent excitation sources.
Remarkably, in modern flow cytometers DRAQ5™ can be efficiently excited by blue through red lasers and is detected in a channel similar to those described above for microscopy.
On the Imagestream®x (Merck) imaging flow cytometer DRAQ5™ is detected in channel 11 (Camera 2) or channel 5 (Camera 1).
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However, if your microscope only has blue laser excitation or cannot detect far-red emission signals or you've got another red/far-red fluorophore in your flow analysis that cannot be compensated for..
However, if your microscope only has blue laser excitation or cannot detect far-red emission signals or you've got another red/far-red fluorophore in your flow analysis that cannot be compensated for..
CyTRAK Orange™ has peak absorbance at 515 nm so is best excited by 488 nm or 532 nm sources. CyTRAK Orange™'s peak emission is centred on 615 nm so a filter/channel previously designated for propidium iodide is ideal.
Here then you can excite GFP and CYTRAK Orange™ simultaneously off the same excitation source for ideal pixel-pixel registration and without needing a UV-source or far-red detection - meaning a simple wide-field fluorescence microscope should be sufficient.
For flow cytometry, CyTRAK Orange™ can be excited by the blue laser line (488 nm) and detected in the channel otherwise specified for propidium iodide.
Here then you can excite GFP and CYTRAK Orange™ simultaneously off the same excitation source for ideal pixel-pixel registration and without needing a UV-source or far-red detection - meaning a simple wide-field fluorescence microscope should be sufficient.
For flow cytometry, CyTRAK Orange™ can be excited by the blue laser line (488 nm) and detected in the channel otherwise specified for propidium iodide.
On the Imagestream®x imaging flow cytometer we recommend detecting CyTRAK Orange™ in channel 4 (Camera 1).
AND, as added bonus..
You can use these cell permeant probes for your fixed cell preps and FFPE tissue sections as well, already cited in thousands of publications.
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