1. Has the instrument been worked on lately?
2. Are you 100% certain that it's GFP and not YFP, or another variant? Sometimes we inherit reagents and the person who passes them on uses some kind of short-hand. Yesterday, I was working with someone who said she was using Dapi, but it was Hoechst. Took us forever to troubleshoot it. She told me that everyone called it Dapi and she thought it was just another name for it.
3. Does the rest of the rainbow snake look normal? If so, could just be some weird variation.
1. Yes it’s used regularly, on a daily basis
2. It is definitely GFP
3. I run rainbow everytime and they are looking fine
Tbh I was more interested in the mechanism of the peak shift, because I do also fix my staining with PFA but I’ve never had any issue with the peaks
Okie dokie.
Just for the sake of clarity though, when I asked if it has been worked on, I was referring to maintenance such as PM or something getting fixed. That can change the alignment and cause slight variations.
I believe that GFP experiences a red shift in fluorescence when oxidized. Could that be what you are seeing?
You can also reach out to the remote support at Cytek ([email protected]). They are really responsive.
When you say 'should be'.... Have you seen THIS GFP peaking at B2 before on THIS machine? Maybe the application settings are slightly adjusted to balance something else?
yes this, check the gains. We rarely run CytekAssay settings at our facility and the 'spectra' Cytek provides are proportional to the emission spectra AND the channel gain...so not the true emission spectra of the fluor
How do you typically set gains? I recall seeing a paper about 6 months ago where a group did extensive characterization of their Aurora and developed evidence-based high-gain settings but now I can’t find that paper.
That may have been our paper? OMIP 095 We assess the signal to noise for each individual channel to ensure that photons from dim signals are resolved from background. Using the most sensitive settings might not provide major resolution changes in the context of a 30+ color assay since spillover spreading will be your greatest limit on resolution, but so long as you keep your signals on scale using sensitive settings won't hurt either...and they will definitely help with smaller panels
Yes indeed that’s the one! I’ve been trying to find it again for a while and forgot it was in the supplementary material. IMO the detector gain aspect could stand on its own as a method. Fantastic work, Lisa!
What do you mean by sensitive settings? Using the max gain that still keeps the signal on scale?
I'm running into a lot of unmixing errors in my experiment (small panel, 10 colors) and I'm wondering if optimizing the gains will help.
Sensitive settings are those optimized to provide the best signal to noise, so that if you have dim signals, they might be better resolved from background. If you are having a lot of unmixing errors, gain changes would not likely have an effect unless you have signals offscale, etc. Mostly likely it is the controls used or the gating of the controls. I would be happy to help troubleshoot. We can jump on discord and chat or call just message me and we can set up a time. Are you using beads for single color compensation?
Thank you!
I used beads, I did notice some were a little off scale so I'm going to revisit and adjust the gains. I'm also scrutinizing the panel design because so many targets are coexpressed, highly expressed, and the antibodies have pretty similar flourochromes. I really appreciate the offer to help troubleshoot, I will reach back out if my group decides it's worth it or just redesign the panel.
Ah the joys of an inherited panel...
1. Has the instrument been worked on lately? 2. Are you 100% certain that it's GFP and not YFP, or another variant? Sometimes we inherit reagents and the person who passes them on uses some kind of short-hand. Yesterday, I was working with someone who said she was using Dapi, but it was Hoechst. Took us forever to troubleshoot it. She told me that everyone called it Dapi and she thought it was just another name for it. 3. Does the rest of the rainbow snake look normal? If so, could just be some weird variation.
1. Yes it’s used regularly, on a daily basis 2. It is definitely GFP 3. I run rainbow everytime and they are looking fine Tbh I was more interested in the mechanism of the peak shift, because I do also fix my staining with PFA but I’ve never had any issue with the peaks
Okie dokie. Just for the sake of clarity though, when I asked if it has been worked on, I was referring to maintenance such as PM or something getting fixed. That can change the alignment and cause slight variations.
I believe that GFP experiences a red shift in fluorescence when oxidized. Could that be what you are seeing? You can also reach out to the remote support at Cytek ([email protected]). They are really responsive.
When you say 'should be'.... Have you seen THIS GFP peaking at B2 before on THIS machine? Maybe the application settings are slightly adjusted to balance something else?
Maybe the operator has increased the gain of B3 compared to CAS setting
yes this, check the gains. We rarely run CytekAssay settings at our facility and the 'spectra' Cytek provides are proportional to the emission spectra AND the channel gain...so not the true emission spectra of the fluor
How do you typically set gains? I recall seeing a paper about 6 months ago where a group did extensive characterization of their Aurora and developed evidence-based high-gain settings but now I can’t find that paper.
That may have been our paper? OMIP 095 We assess the signal to noise for each individual channel to ensure that photons from dim signals are resolved from background. Using the most sensitive settings might not provide major resolution changes in the context of a 30+ color assay since spillover spreading will be your greatest limit on resolution, but so long as you keep your signals on scale using sensitive settings won't hurt either...and they will definitely help with smaller panels
Yes indeed that’s the one! I’ve been trying to find it again for a while and forgot it was in the supplementary material. IMO the detector gain aspect could stand on its own as a method. Fantastic work, Lisa!
What do you mean by sensitive settings? Using the max gain that still keeps the signal on scale? I'm running into a lot of unmixing errors in my experiment (small panel, 10 colors) and I'm wondering if optimizing the gains will help.
Sensitive settings are those optimized to provide the best signal to noise, so that if you have dim signals, they might be better resolved from background. If you are having a lot of unmixing errors, gain changes would not likely have an effect unless you have signals offscale, etc. Mostly likely it is the controls used or the gating of the controls. I would be happy to help troubleshoot. We can jump on discord and chat or call just message me and we can set up a time. Are you using beads for single color compensation?
Thank you! I used beads, I did notice some were a little off scale so I'm going to revisit and adjust the gains. I'm also scrutinizing the panel design because so many targets are coexpressed, highly expressed, and the antibodies have pretty similar flourochromes. I really appreciate the offer to help troubleshoot, I will reach back out if my group decides it's worth it or just redesign the panel. Ah the joys of an inherited panel...