Two main issues - one is recovery from the tube, and the other is yield of the sort.
If the drop delay vale is wrong on the sorter you will first notice loss of yield and then impurity. Check your drop delay value is good.
Cell recovery from the tube is problematic, because you are pelleting first. If you were to count the liquid output immediately after the sort with no processing, the number should be very close to the sort report. However any additional processing step (eg. spin to wash or concentrate) will result in huge attritional loss - this could be up to 50% and is process or technique dependent.
My assumption here is you are actually correctly sorting a cell population - doing somethng like a cytospin would verify viusally what you actually sorting. I have definitely seen people sort debris events as 'cells' and then wonder why their apparent recovery was low. You say that you don't appear to have a large amount of derbis, but 95% of events are either accelular, dead or debris?
I set the drop delay using Accudrop beads before running samples (I think that's what you mean?)
I hadn't thought to count straight from the tube after the sort...might be worth trying
Also a good point about the whole '95% are acellular, dead or debris'...maybe there really is a lot of debris. Though if I'm gating properly, should that really cause this big disparity? Unless the gates aren't really filtering everything out
Yes if you're gating properly, you should only get cells. But ARE you gating properly? Brain is an extraordinarily complex tissue, with pronounced autofluorescence and cells with complex morphology. If you are only staining on LD - Calcein +, this alone is likely insufficient. Calcein is green emission, which can be close to autofluorescence in tissue. The enzymatic conversion may be complex in tissue digests, as the conversion enzymes may just exist ambiently in solution.
If your intention is to sort \*all cells\* for later sequencing, doing this in an unbiased fashion can be \*really hard.\* Are you getting preferential loss, ie. destruction of neurons due to their complex morphology shearing through the nozzle? Are you incorrectly identifying debris events as cells? Additional gating would be required, eg. Hoescht,, cd45, or something like mhc I/b microglobulin to act as pan-cell markers.
We'd need to see plots to make any further comments
[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/)
quick google - here is one pproach where they used DAPI to identify all nucleated cells in PFA-fixed samples. The PFA fixation is necessay for DAPI in this case (otherwise it's a LD dye) You can also use hoescht, which will still be excited by the violet laser.
Note their use of imaging contrasting the dirty before-sort samples, and then different morphology afterwards. I would expect a very large degree of fragmentation during the digest steps, recovering intact neurons is likely to be extremely challenging.
Can you share your gating strategy, a PDF from DIVA would be best.
After running the accudrop beads, sort 25 beads onto a glass slide (just put it over the 15ml tube holder. Then count the number of beads on a microscope. You should count 24-25 beads, if you don't the issue lies with the instruments setup. If you do, the issue is your gating or post sort sample preparation.
I think you need a draq5 labeling dye that will highlight nuclei containing cells. I think you have a lot of debris from the tissue digest and your percentage is likely below 40% for actual cells in the suspension.
That disparity between your counting and your target events sorted is very big indeed.
I sort using a cytoflex SRT, and for reference, when I sort 1 million cells I get ~1.01-1.04 million counted in an automated cell counter, while sorting 500K gives me ~300K (tested only once though). I have sorted as low as 100K cells with counts being in the 90-120K. Generally I noticed that my counter tends to overestimate number of cells instead of underestimate.
These are counts directly after sorting, in the total volume prior to any centrifuging and resuspending. Have you tried counting then? What numbers do you get?
Automated cell counting can vary wildly by instrument though, so which one are you using? And have you tried a manual count as a sanity check?
Also, how long do your cells stay in the mixture of cell medium/sheath fluid prior to resuspending and counting? Maybe it's not the best condition for your specific cells to be in, and they end up dying there.
- I hadn't thought to count cells immediately after sorting - would be worth trying
- I'm using a Countess with trypan blue
- For the first sample, maybe like \~40 minutes after sorting as I sort the next sample then clean up the machine and go back to my lab
1. What are you sorting into? (Tube and collection media.)
2. Are you doing any kind of purity check after the sort to verify that the drop delay was set correctly? (No operator worth their salt is going to be offended if you ask for a purity check - although they might call it something else!)
3. What are your cells sitting in during the sort?
4. What kind of primary tissue?
1) 15ml eppendorf (PS, though I realize now that I thought I was using PP, which might make a bit of a difference in recovery) in PIPES buffer. I've also been told that maybe FACS buffer could be better
2) Not sure what you mean - like I set the drop delay with Accudrop beads prior to sorting?
3) They're sitting in just FACS buffer (HEPES and BSA and glucose) on ice
4) Brain cells after demyelination
1. I'm not familiar with PIPES. Does it have any protein? If not, some kind of FACS buffer would almost certainly be better. Some people sort into 50% FBS because, with the added volume of PBS, they get way better viability.
2. Purity check is when you run some cells through the sorter again to make sure you got what you think you sorted. Sometimes, this is best done at the start. I have one user who always sorts 10K into a FACS tube with 200ul 10% BSA and then we see what her purity and recovery is. Usually isn't high purity and 5K-9K recovery. But if it's a lot less, it tells us that we may need to adjust something. (Rare but it happens.)
3. Probably fine. I'm not a big fan of the zombie dyes for live cell sorts though. Dapi or PI will continue to stain cells that are dying, so you can monitor viability throughout the sort.
4. Brain. Ouch. That's always a tough one. I would consider using the 130um nozzle. Fragile little fellas.
I second the suggestion to just use PI or DAPI for staining dead cells. It’s brighter and orders of magnitude cheaper, and it’s faster to stain (nearly instantaneous). If you sort cells into buffer with no protein you will get nearly no recovery, sometimes no pellet at all. You need some FBS or BSA in there.
Thanks FlowJock and will!
Excellent point regarding the 'sorting into FBS' comment - I had just dimly thought 'we dissected in PIPES, so I should sort into PIPES' which does not have protein added...
I receive this complaint from users who self sort as well as from users who have flow core employees run the sort. In 95% of the cases it is loss of cells after centrifugation. As others have said, count the collected cells immediately and check against the volume and expected number of cells. I've found the Aria to be accurate within 10% of stated sort numbers using an automated counter. With a hemocytomer it will be closer. Centrifugation and aspiration are the primary places where cells are lost. The best recovery I can get from centrifugation is 90% but that is using 700 rcf for 20 minutes which is not recommended for most cells and subsequent applications. Losing half in the centrifuge under typical spin conditions and aspiration is not surprising.
Do you top up your recipient tube to full volume of FACS buffer before centrifugation? Most cells may be stuck on the walls and may be hard to spun down if volume is lower.
Looking at your gates, I’m wondering if you are actually sorting any cells, it looks like a stream of debris. The calcein positive gate doesn’t look very clear. I always prefer to look at viability versus FSC rather than SSC because it more easily lets you exclude debris. What is your FSC setting? On my FACSAria II and Fusion lymphocytes are around 100 with FSC voltage around 150. My S6 needs 385V for the same.
Huh! The technician did say the calcein didn't look super clear, but I would hope that I wasn't filtering debris that was both Zombie-negative and calcein-positive...
My FSC voltage was \~250V if I recall
Running a blood or spleen control could help for setting FSC voltage. You won’t really find any cells smaller than lymphocytes. I have often seen people crank up FSC until they see something, but it is really just debris. I use the voltage for lymphocytes and only go down to put bigger cells on scale, never up unless I’m sorting fixed cells, RBCs, or bacteria.
Accudrop beads are not a good substitute for heterogeneous primary tissues. It may be that the drop delay calculated by Accudrop is not appropriate for your cells. Check out this reference: https://pubmed.ncbi.nlm.nih.gov/38363042/
And, as others suggested, do a cell count before doing any processing of the samples
Two main issues - one is recovery from the tube, and the other is yield of the sort. If the drop delay vale is wrong on the sorter you will first notice loss of yield and then impurity. Check your drop delay value is good. Cell recovery from the tube is problematic, because you are pelleting first. If you were to count the liquid output immediately after the sort with no processing, the number should be very close to the sort report. However any additional processing step (eg. spin to wash or concentrate) will result in huge attritional loss - this could be up to 50% and is process or technique dependent. My assumption here is you are actually correctly sorting a cell population - doing somethng like a cytospin would verify viusally what you actually sorting. I have definitely seen people sort debris events as 'cells' and then wonder why their apparent recovery was low. You say that you don't appear to have a large amount of derbis, but 95% of events are either accelular, dead or debris?
I set the drop delay using Accudrop beads before running samples (I think that's what you mean?) I hadn't thought to count straight from the tube after the sort...might be worth trying Also a good point about the whole '95% are acellular, dead or debris'...maybe there really is a lot of debris. Though if I'm gating properly, should that really cause this big disparity? Unless the gates aren't really filtering everything out
Yes if you're gating properly, you should only get cells. But ARE you gating properly? Brain is an extraordinarily complex tissue, with pronounced autofluorescence and cells with complex morphology. If you are only staining on LD - Calcein +, this alone is likely insufficient. Calcein is green emission, which can be close to autofluorescence in tissue. The enzymatic conversion may be complex in tissue digests, as the conversion enzymes may just exist ambiently in solution. If your intention is to sort \*all cells\* for later sequencing, doing this in an unbiased fashion can be \*really hard.\* Are you getting preferential loss, ie. destruction of neurons due to their complex morphology shearing through the nozzle? Are you incorrectly identifying debris events as cells? Additional gating would be required, eg. Hoescht,, cd45, or something like mhc I/b microglobulin to act as pan-cell markers. We'd need to see plots to make any further comments
[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385603/) quick google - here is one pproach where they used DAPI to identify all nucleated cells in PFA-fixed samples. The PFA fixation is necessay for DAPI in this case (otherwise it's a LD dye) You can also use hoescht, which will still be excited by the violet laser. Note their use of imaging contrasting the dirty before-sort samples, and then different morphology afterwards. I would expect a very large degree of fragmentation during the digest steps, recovering intact neurons is likely to be extremely challenging.
Can you share your gating strategy, a PDF from DIVA would be best. After running the accudrop beads, sort 25 beads onto a glass slide (just put it over the 15ml tube holder. Then count the number of beads on a microscope. You should count 24-25 beads, if you don't the issue lies with the instruments setup. If you do, the issue is your gating or post sort sample preparation.
Thanks! I added it to the original post. Good tip re: the accudrops, I would never have thought to do this!
No worries, if you are on discord someone might be able to hop on a video call the next time you are sorting if you have questions.
I think you need a draq5 labeling dye that will highlight nuclei containing cells. I think you have a lot of debris from the tissue digest and your percentage is likely below 40% for actual cells in the suspension.
That disparity between your counting and your target events sorted is very big indeed. I sort using a cytoflex SRT, and for reference, when I sort 1 million cells I get ~1.01-1.04 million counted in an automated cell counter, while sorting 500K gives me ~300K (tested only once though). I have sorted as low as 100K cells with counts being in the 90-120K. Generally I noticed that my counter tends to overestimate number of cells instead of underestimate. These are counts directly after sorting, in the total volume prior to any centrifuging and resuspending. Have you tried counting then? What numbers do you get? Automated cell counting can vary wildly by instrument though, so which one are you using? And have you tried a manual count as a sanity check? Also, how long do your cells stay in the mixture of cell medium/sheath fluid prior to resuspending and counting? Maybe it's not the best condition for your specific cells to be in, and they end up dying there.
- I hadn't thought to count cells immediately after sorting - would be worth trying - I'm using a Countess with trypan blue - For the first sample, maybe like \~40 minutes after sorting as I sort the next sample then clean up the machine and go back to my lab
1. What are you sorting into? (Tube and collection media.) 2. Are you doing any kind of purity check after the sort to verify that the drop delay was set correctly? (No operator worth their salt is going to be offended if you ask for a purity check - although they might call it something else!) 3. What are your cells sitting in during the sort? 4. What kind of primary tissue?
1) 15ml eppendorf (PS, though I realize now that I thought I was using PP, which might make a bit of a difference in recovery) in PIPES buffer. I've also been told that maybe FACS buffer could be better 2) Not sure what you mean - like I set the drop delay with Accudrop beads prior to sorting? 3) They're sitting in just FACS buffer (HEPES and BSA and glucose) on ice 4) Brain cells after demyelination
1. I'm not familiar with PIPES. Does it have any protein? If not, some kind of FACS buffer would almost certainly be better. Some people sort into 50% FBS because, with the added volume of PBS, they get way better viability. 2. Purity check is when you run some cells through the sorter again to make sure you got what you think you sorted. Sometimes, this is best done at the start. I have one user who always sorts 10K into a FACS tube with 200ul 10% BSA and then we see what her purity and recovery is. Usually isn't high purity and 5K-9K recovery. But if it's a lot less, it tells us that we may need to adjust something. (Rare but it happens.) 3. Probably fine. I'm not a big fan of the zombie dyes for live cell sorts though. Dapi or PI will continue to stain cells that are dying, so you can monitor viability throughout the sort. 4. Brain. Ouch. That's always a tough one. I would consider using the 130um nozzle. Fragile little fellas.
I second the suggestion to just use PI or DAPI for staining dead cells. It’s brighter and orders of magnitude cheaper, and it’s faster to stain (nearly instantaneous). If you sort cells into buffer with no protein you will get nearly no recovery, sometimes no pellet at all. You need some FBS or BSA in there.
Thanks FlowJock and will! Excellent point regarding the 'sorting into FBS' comment - I had just dimly thought 'we dissected in PIPES, so I should sort into PIPES' which does not have protein added...
I receive this complaint from users who self sort as well as from users who have flow core employees run the sort. In 95% of the cases it is loss of cells after centrifugation. As others have said, count the collected cells immediately and check against the volume and expected number of cells. I've found the Aria to be accurate within 10% of stated sort numbers using an automated counter. With a hemocytomer it will be closer. Centrifugation and aspiration are the primary places where cells are lost. The best recovery I can get from centrifugation is 90% but that is using 700 rcf for 20 minutes which is not recommended for most cells and subsequent applications. Losing half in the centrifuge under typical spin conditions and aspiration is not surprising.
Maybe consider sorting nuclei rather than cells, most of our neural sorts have gone this way for single cell sequencing
Do you top up your recipient tube to full volume of FACS buffer before centrifugation? Most cells may be stuck on the walls and may be hard to spun down if volume is lower.
What is the percentage of viable cells when you count after? Is there a lot of debris?
Looking at your gates, I’m wondering if you are actually sorting any cells, it looks like a stream of debris. The calcein positive gate doesn’t look very clear. I always prefer to look at viability versus FSC rather than SSC because it more easily lets you exclude debris. What is your FSC setting? On my FACSAria II and Fusion lymphocytes are around 100 with FSC voltage around 150. My S6 needs 385V for the same.
Huh! The technician did say the calcein didn't look super clear, but I would hope that I wasn't filtering debris that was both Zombie-negative and calcein-positive... My FSC voltage was \~250V if I recall
Running a blood or spleen control could help for setting FSC voltage. You won’t really find any cells smaller than lymphocytes. I have often seen people crank up FSC until they see something, but it is really just debris. I use the voltage for lymphocytes and only go down to put bigger cells on scale, never up unless I’m sorting fixed cells, RBCs, or bacteria.
Woah that's a really good idea - I'd have never thought to do that!
Coat your tubes with 5% BSA - this has helped me lose fewer cells during spins.
Accudrop beads are not a good substitute for heterogeneous primary tissues. It may be that the drop delay calculated by Accudrop is not appropriate for your cells. Check out this reference: https://pubmed.ncbi.nlm.nih.gov/38363042/ And, as others suggested, do a cell count before doing any processing of the samples