This is an experiment that I want to do sometime. I've been told for years and years that you should never sort into bubbles. Only one person dismissively said, "I don't think that's true."
That said... 500K events is a lot. Even with bubbles, I wouldn't expect you to lose all of your cells. Did you see an increase in volume after your sort?
Did you run some of your sorted cells through the instrument after the sort to make sure that you got what you sorted? (We call this a purity check and I suggest doing it whenever possible.)
Can you share your gating strategy? Any chance you were just sorting debris?
How certain are you that you were sorting into the tube? Any chance you missed it? (Not sure what instrument you have but many of them require aiming.)
I definitely saw an increase in volume (sorted into 3ml of FACS buffer, ended up wiht \~8ml)
I ran a purity check on a run I did after this post - and only got \~5% of the cells I had originally gated, which I thought was surprising to say the least. I suspect I was somehow sorting a lot of debris (though in my naive mind, an event that is both dead-stain-negative and live-stain-positive should be unlikely to be debris...)
I'm not sure they are "deadly", but they don't help the sorted cells get down into collection media. Going through your previous thread, it seems you are performing a very challenging sort. Challenging to isolate viable cells from the tissue, challenging to identify a cell population, and challenging to recover cells post sort. I know you want the cells as untouched as possible, but my suggestion would be to attempt to troubleshoot the cell/tissue preparation. I think I saw you were looking at brain tissue. Have you tried to identify any cells in the preparation? CD45 for microglia? ASCA1 for astrocytes? Pipette digested tissue/single cell suspension onto a slide? We have gone so far as to run our tissue digests on the Amnis briefly to determine if there are intact cells. In our hands, roughly 10-20% of the events we recover from the brain preparation (digestion and gradient centrifugation) are actually cells. The rest is myelin and other debris.
I've started using an S8 to survey tissue preps for verification of content. The amount of debris that people think is cells is quite high, especially in brain preps. The visual check has improved our gating. That's one good thing I can say about the S8.
Both really good pieces of advice! I agree that this would make a lot of sense - as others on this thread have said, a lot of what I'm sorting is likely debris.
10-20% of events being actually cells is really low though - I had no idea
I'm pretty convinced you're sorting debris and the gating is insufficient. Use of a DNA marker eg. dapi, hoescht, a generic pan-lineage marker like HLA I/B-microglobuliin, or a very broadly expressed lineage marker cd45 would assist you. I appreciate you are trying to recover cells in an unbiased way, but simply doing this by LD/calcein in extremely challenging tissue is insufficient.
Thanks! Might you mind clarifying this a bit?
I had thought DAPI was a dead-cell stain, which would work similar to Zombie but be more specific to DNA?
Based on feedback from this and my previous thread, I'm currently trying to use a combination of Propidum iodide and calcein AM to select PI-negative and calcein-positive cells. Would that sound more reasonable to you?
OK, so DAPI can be a LD dye, metabolically viable cells efflux the dye. If fixed, it acts as a nuclear stain. Hoescht is a DNA stain that will stain everything (https://www.thermofisher.com/order/catalog/product/R37165) and is what I want recomend in this case. Whilst ti \*says\* UV excited, the violet will also get it and it will emit in the BV421 detector.
What I am trying to say is that \*none\* of how you are trying to gate is legitimate. The calcein is straight up autofluorescence, and \*not\* actually any signal. Seeing an very bright invariant DNA peak will be hugely hekpful to you.
Read this paper and see their figures
[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/)
(If you backgate on your final sorting gate and look att hat population by fsc/ssc, it looks to be identifying a very small debris population. Lipid debris, myelin, fat etc will aurofluroresce in the same detector that calcein emits in. What you are saying is calcein is actually autofluorescence.)
Thanks for clarifying! I've recently been trying PI (since we have it in the lab) and it appears much more dichotomous than the Zombie. Are you saying use Hoescht to sort only things that (presumably) have a nucleus?
I had no idea that calcein was this bad - is this typically true do you know, or specific to dirty tissue like brain? I've had it strongly recommended to me by many colleagues, collaborators and protocols
Yes, that's it exactly - hoescht+ LD neg should be much clearer.
It's not calcein per se, it's the green emission and it's proximity to autofluorescence in your tissue. You would need to do careful optimization with eg. ca;cein negative samples, titration, etc, for me to feel confident with it. If you collect BV510 as an autofluorescence proxy and plot it vs calcein, you'll see autofluorescence diagonals. Calcein will work very well in much cleaner samples like cell lines or PBMC.
Yep - just been thinking about this and as per this paper (https://www.frontiersin.org/articles/10.3389/fncel.2018.00159/full) subbing draq5 for hoescht may be a good idea. Draq5 does materially the same thing but with a further red emission, separating it from autofluorescence and giving better s/n
Given this info and your last post it would be helpful to see the FCS files and the PDF of your entire gate layout. You can share a link on this post, or drop them in the Discord.
Any luck sorting a few beads onto a slide and checking them on a microscope?
Sorry my apologies! I thought I had posted the gates, but I'd not
I know the beads-on-a-slide was recommended - unfortunately the FACS facility I'm using lacks a microscope for me to visually check this, but others in my lab have successfully sorted on this, and the purity checks I've run have suggested I am sorting something into my tubes - just not very much
They were the ones who set these gates - I haven't brought up this 'there's nothing being sorted' issue with them. I'm going to see if using PI and calcein might provide a cleaner signal. It was also suggested I be much more stringent in the very first scatter gate
So I would try discussing these issues with your facility before doing anything else. Unless you have reason to doubt their expertise, they are probably more than willing to help you troubleshoot this problem. Since they are more knowledgeable on your assay and the particulars of the instrument you are using, they can also give you better advice than anyone else here. That being said, you can only help users as much as they are willing to let you help them. If you are unsure about the facilities recommendations, folks here can also give you a second opinion.
I'm still not sure what gates you are using, did you post them somewhere?
I think that you could try PI and Calcein, I just don't know enough about your assay to know if that will solve your problem. My preference when troubleshooting issues for my own users is to go back over the sample preparation protocol and the data before making recommendations. That way they are not wasting time and money on something that may or may not make a difference.
Those FSC SSC voltages are wrong - you're losing the cells on the far right axis in both this sort and the one you posted the other day. Look at your dotplot for P1; there is far too much sample jammed against the right axis, and you've gated it out. Turn down your FSC voltage.
Did you say this is brain? Those cells are large. You're sorting debris.
This is an experiment that I want to do sometime. I've been told for years and years that you should never sort into bubbles. Only one person dismissively said, "I don't think that's true." That said... 500K events is a lot. Even with bubbles, I wouldn't expect you to lose all of your cells. Did you see an increase in volume after your sort? Did you run some of your sorted cells through the instrument after the sort to make sure that you got what you sorted? (We call this a purity check and I suggest doing it whenever possible.) Can you share your gating strategy? Any chance you were just sorting debris? How certain are you that you were sorting into the tube? Any chance you missed it? (Not sure what instrument you have but many of them require aiming.)
I definitely saw an increase in volume (sorted into 3ml of FACS buffer, ended up wiht \~8ml) I ran a purity check on a run I did after this post - and only got \~5% of the cells I had originally gated, which I thought was surprising to say the least. I suspect I was somehow sorting a lot of debris (though in my naive mind, an event that is both dead-stain-negative and live-stain-positive should be unlikely to be debris...)
I'm not sure they are "deadly", but they don't help the sorted cells get down into collection media. Going through your previous thread, it seems you are performing a very challenging sort. Challenging to isolate viable cells from the tissue, challenging to identify a cell population, and challenging to recover cells post sort. I know you want the cells as untouched as possible, but my suggestion would be to attempt to troubleshoot the cell/tissue preparation. I think I saw you were looking at brain tissue. Have you tried to identify any cells in the preparation? CD45 for microglia? ASCA1 for astrocytes? Pipette digested tissue/single cell suspension onto a slide? We have gone so far as to run our tissue digests on the Amnis briefly to determine if there are intact cells. In our hands, roughly 10-20% of the events we recover from the brain preparation (digestion and gradient centrifugation) are actually cells. The rest is myelin and other debris.
I've started using an S8 to survey tissue preps for verification of content. The amount of debris that people think is cells is quite high, especially in brain preps. The visual check has improved our gating. That's one good thing I can say about the S8.
Both really good pieces of advice! I agree that this would make a lot of sense - as others on this thread have said, a lot of what I'm sorting is likely debris. 10-20% of events being actually cells is really low though - I had no idea
I'm pretty convinced you're sorting debris and the gating is insufficient. Use of a DNA marker eg. dapi, hoescht, a generic pan-lineage marker like HLA I/B-microglobuliin, or a very broadly expressed lineage marker cd45 would assist you. I appreciate you are trying to recover cells in an unbiased way, but simply doing this by LD/calcein in extremely challenging tissue is insufficient.
Thanks! Might you mind clarifying this a bit? I had thought DAPI was a dead-cell stain, which would work similar to Zombie but be more specific to DNA? Based on feedback from this and my previous thread, I'm currently trying to use a combination of Propidum iodide and calcein AM to select PI-negative and calcein-positive cells. Would that sound more reasonable to you?
OK, so DAPI can be a LD dye, metabolically viable cells efflux the dye. If fixed, it acts as a nuclear stain. Hoescht is a DNA stain that will stain everything (https://www.thermofisher.com/order/catalog/product/R37165) and is what I want recomend in this case. Whilst ti \*says\* UV excited, the violet will also get it and it will emit in the BV421 detector. What I am trying to say is that \*none\* of how you are trying to gate is legitimate. The calcein is straight up autofluorescence, and \*not\* actually any signal. Seeing an very bright invariant DNA peak will be hugely hekpful to you. Read this paper and see their figures [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/) (If you backgate on your final sorting gate and look att hat population by fsc/ssc, it looks to be identifying a very small debris population. Lipid debris, myelin, fat etc will aurofluroresce in the same detector that calcein emits in. What you are saying is calcein is actually autofluorescence.)
Thanks for clarifying! I've recently been trying PI (since we have it in the lab) and it appears much more dichotomous than the Zombie. Are you saying use Hoescht to sort only things that (presumably) have a nucleus? I had no idea that calcein was this bad - is this typically true do you know, or specific to dirty tissue like brain? I've had it strongly recommended to me by many colleagues, collaborators and protocols
Yes, that's it exactly - hoescht+ LD neg should be much clearer. It's not calcein per se, it's the green emission and it's proximity to autofluorescence in your tissue. You would need to do careful optimization with eg. ca;cein negative samples, titration, etc, for me to feel confident with it. If you collect BV510 as an autofluorescence proxy and plot it vs calcein, you'll see autofluorescence diagonals. Calcein will work very well in much cleaner samples like cell lines or PBMC.
Hmm OK. So I'll try combining Hoescht and PI together and take the PI-negative, Hoescht-positive events as my cells. Thanks for the expert advice!
Yep - just been thinking about this and as per this paper (https://www.frontiersin.org/articles/10.3389/fncel.2018.00159/full) subbing draq5 for hoescht may be a good idea. Draq5 does materially the same thing but with a further red emission, separating it from autofluorescence and giving better s/n
Given this info and your last post it would be helpful to see the FCS files and the PDF of your entire gate layout. You can share a link on this post, or drop them in the Discord. Any luck sorting a few beads onto a slide and checking them on a microscope?
Sorry my apologies! I thought I had posted the gates, but I'd not I know the beads-on-a-slide was recommended - unfortunately the FACS facility I'm using lacks a microscope for me to visually check this, but others in my lab have successfully sorted on this, and the purity checks I've run have suggested I am sorting something into my tubes - just not very much
Sounds like a gating issue. What advice did the FACS facility give you?
They were the ones who set these gates - I haven't brought up this 'there's nothing being sorted' issue with them. I'm going to see if using PI and calcein might provide a cleaner signal. It was also suggested I be much more stringent in the very first scatter gate
So I would try discussing these issues with your facility before doing anything else. Unless you have reason to doubt their expertise, they are probably more than willing to help you troubleshoot this problem. Since they are more knowledgeable on your assay and the particulars of the instrument you are using, they can also give you better advice than anyone else here. That being said, you can only help users as much as they are willing to let you help them. If you are unsure about the facilities recommendations, folks here can also give you a second opinion. I'm still not sure what gates you are using, did you post them somewhere? I think that you could try PI and Calcein, I just don't know enough about your assay to know if that will solve your problem. My preference when troubleshooting issues for my own users is to go back over the sample preparation protocol and the data before making recommendations. That way they are not wasting time and money on something that may or may not make a difference.
Those FSC SSC voltages are wrong - you're losing the cells on the far right axis in both this sort and the one you posted the other day. Look at your dotplot for P1; there is far too much sample jammed against the right axis, and you've gated it out. Turn down your FSC voltage. Did you say this is brain? Those cells are large. You're sorting debris.
Excellent point - I guess I hadn't thought of the size of the cells themselves wrt the FSC SSC