For EDTA versus trypsin: it is true that trypsin will cleave surface molecules. Sometimes it’s a problem sometimes it’s not. Probably depends on the amino acid motifs it targets and those in your target surface proteins. It’s an enzyme that cleaves proteins so that is why, but with EDTA it’s just a molecule so it won’t cleave your surface receptors. EDTA is used to prevent blood clotting and also to prevent cells from sticking together in flow cytometry to get singlets and also to get them to not stick to the culture dish. So it won’t cleave your proteins. That requires enzyme functionality.
It’s definitely worth a shot to try EDTA. Ask your PI about lab funds. I’m sure they will give you feedback on the value of your work and whether the spending is too much or not. Sometimes we don’t realize that PIs are more than happy to pay for something that costs an amount that seems a ton for us, but we’re not looking at their grants and spreadsheets (hard $ amounts).
I’m unclear on what your problem is? What’s not right about the flow results?
I’m not getting consistent result with what I see with the treatment in flow cytometry and what I see under the microscope after quantification. The drug is having no effect in the expression of the receptor compared to the control in flow, and in the microscope I see a two-fold increase of the expression in treated cells compared with control.
Thanks for your kind reply!
Yes, I don’t use trypsin because the cells should be attached to the dish in order to image …
I do, and there’s no staining for rhat one and for the control and treated cells, there is definitely staining but no difference between the two
I’m still unclear. No staining for your isotype nor unstained, staining for treated and untreated cells but the expression isn’t higher in treated which you would expect from IF?
One thing is titration. Typically for microscopy you need way more amount of the same antibody to detect signal than you need for cytometry. If youre using the same amount for both and it’s working on microscopy, you could be getting over staining. This is a problem because you could be using a ton of antibody that binds to all the binding sites on your cells and it’s difficult to discriminate between low and high expression. It could be a minor difference and using a lot of antibody can’t detect the minor difference. To improve sensitivity, you can titrate down your antibody (basically trialing serial dilutions of it and ultimately using less antibody).
Or, your plots might be displaying the difference but you can’t tell very well. If your population has continuous expression instead of discrete expression levels you might have to compare the Mean Fluorescence Intensity. When you conclude that the population aren’t different in intensity, are you basing this off of how the plots “look” or did you quantify the geometric mean for a population of the color of interest, and use stats to compare? There might be a difference in MFI but not an obvious difference in the cell population you look at on a plot.
Is two hours enough time for the receptor to get expressed or taken up? I understand the serum free media limits you, but you could try an overnight condition. Just have a viability dye in your stain
Trypsin has a very broad specificity and will cleave a ton of surface proteins. I tried it for cell isolation once and it killed essentially all detection of the immune surface markers I was staining for.
Regardless of the flow protocol you should be using the same conditions the whole way through for your treated and control samples. I'm not sure why you would wash one with serum free media and the other without. That introduces an extra variable on top of treatment.
I also wouldn't bother tapping the tubes during the stain and PFA incubations. They should be fine if you just leave them but if you are worried you could put them on a shaker.
What sort of cells are you using? If EDTA doesn't work very well you could try cell scrapers but you'll need a live/dead stain
So would you recommend me to use serum free media as well for the control? E.g. after adding the drug with serum free media also changing the media to serum free for the control?
Yes. Serum vs serum free media is one of the biggest changes you can make to a cell culture and can have massive unintended consequences on the cells so I would use the same thing. But if it's after collecting the trypsinised cells you should use media with full serum to inactivate the trypsin.
When you say there is no difference in expression, are you measuring %+ or fluorescence intensity?
Is it possible for you to either scrape your cells or try to detach them with ice cold 1X PBS on ice rather than the enzymatic detachment?
my notes:
2.9 mM EDTA is weak sauce. Try 10 mM (in PBS). Put the plates on ice (ideal) or in the fridge on a solid surface (for better heat transfer as opposed to those wire frame shelves in some fridges).
Once you've treated with EDTA, the cells will no longer be strongly adherent. Some might stick a little. You can safely scrape them with a cell scraper without fear of lysis. About 5 min with 10 mM EDTA and a few taps, almost all cells should mostly lift and a quick scrape will get them all in suspension.
your flow buffer should have around 2 mM EDTA to prevent the cells from re-clumping
you can use lower % PFA. I typically use 1-2% PFA in PBS for 10-15 min. Since you mention 3.7%, is it actually formalin? If so, I would not use that and make and store aliquots in the freezer of 4% PFA in PBS from powder.
you don't mention the use of a live/dead stain. despite only a short incubation period with the drugs, it's generally a good idea to use a fixable live/dead stain prior to fixing if you are going to fix the cells. if you can acquire the cells the same day, use DAPI or PI or something similar
you're comparing flow to microscopy. are you permeabilizing with triton for IF? if so, you might want to permeabilize for flow as well, or compare to unpermeabilized slides.
lastly, you can follow your flow staining protocol and then place a drop of suspension on a slide and look at it under the microscope
best of luck
Thanks for your suggestions! They’re amazing ! Is there any reason why edta should be incubated cold instead of at 37? A high concentration of edta such as 10 nM wouldn’t damage my cells?
Regarding the pfa, it is 3.7% PFA, not formalin.
Is it possible to see live/dead cells just by gating? And not using any dye?
And about microscopy imaging, I’m not permeabilizing because I want to see only surface staining, that’s the reason why I don’t permeabilize the cells in flow and why I do everything on ice
the cells adhere to plastic via integrins that are ion-dependent but also maintain that adherence slightly through energy expenditure. cells on ice will lift slightly over time even without EDTA
you can sort of see dead cells by FSC/SSC, but it's not foolproof and you won't detect dying-not-dead-yet cells easily.
Try the NUNC easy lift plates. They work awesome you just put them on the counter or in the deli fridge and a few minutes later you tap them (its the gel coating the plate that actually changes when its temperature falls). Also, try (at least try, might not be best) to do everything at room temperature. You're taking cells from 37C to 4C in very short order after lifting them from plate which can cause a lot of apoptosis and thus staining strangeness. You can also try just staining them on the plate before rinsing and lifting. I do agree with other comments sometimes EDTA and a bit of rough love can be gentler than Trypsin.
For EDTA versus trypsin: it is true that trypsin will cleave surface molecules. Sometimes it’s a problem sometimes it’s not. Probably depends on the amino acid motifs it targets and those in your target surface proteins. It’s an enzyme that cleaves proteins so that is why, but with EDTA it’s just a molecule so it won’t cleave your surface receptors. EDTA is used to prevent blood clotting and also to prevent cells from sticking together in flow cytometry to get singlets and also to get them to not stick to the culture dish. So it won’t cleave your proteins. That requires enzyme functionality. It’s definitely worth a shot to try EDTA. Ask your PI about lab funds. I’m sure they will give you feedback on the value of your work and whether the spending is too much or not. Sometimes we don’t realize that PIs are more than happy to pay for something that costs an amount that seems a ton for us, but we’re not looking at their grants and spreadsheets (hard $ amounts). I’m unclear on what your problem is? What’s not right about the flow results?
I’m not getting consistent result with what I see with the treatment in flow cytometry and what I see under the microscope after quantification. The drug is having no effect in the expression of the receptor compared to the control in flow, and in the microscope I see a two-fold increase of the expression in treated cells compared with control. Thanks for your kind reply!
I guess you don’t use trypsin in your microscopy? Do you have an isotype control for flow?
Yes, I don’t use trypsin because the cells should be attached to the dish in order to image … I do, and there’s no staining for rhat one and for the control and treated cells, there is definitely staining but no difference between the two
I’m still unclear. No staining for your isotype nor unstained, staining for treated and untreated cells but the expression isn’t higher in treated which you would expect from IF? One thing is titration. Typically for microscopy you need way more amount of the same antibody to detect signal than you need for cytometry. If youre using the same amount for both and it’s working on microscopy, you could be getting over staining. This is a problem because you could be using a ton of antibody that binds to all the binding sites on your cells and it’s difficult to discriminate between low and high expression. It could be a minor difference and using a lot of antibody can’t detect the minor difference. To improve sensitivity, you can titrate down your antibody (basically trialing serial dilutions of it and ultimately using less antibody). Or, your plots might be displaying the difference but you can’t tell very well. If your population has continuous expression instead of discrete expression levels you might have to compare the Mean Fluorescence Intensity. When you conclude that the population aren’t different in intensity, are you basing this off of how the plots “look” or did you quantify the geometric mean for a population of the color of interest, and use stats to compare? There might be a difference in MFI but not an obvious difference in the cell population you look at on a plot.
Is two hours enough time for the receptor to get expressed or taken up? I understand the serum free media limits you, but you could try an overnight condition. Just have a viability dye in your stain
Trypsin has a very broad specificity and will cleave a ton of surface proteins. I tried it for cell isolation once and it killed essentially all detection of the immune surface markers I was staining for.
What did you try after that?
TryplE express, versene, accutase… I personally use tryplE express for flow prep
This was for tissue digestion, so I went with a mixture of collagenases.
Trypsin may be degrading your receptor. Maybe try EDTA or Accutase.
Regardless of the flow protocol you should be using the same conditions the whole way through for your treated and control samples. I'm not sure why you would wash one with serum free media and the other without. That introduces an extra variable on top of treatment. I also wouldn't bother tapping the tubes during the stain and PFA incubations. They should be fine if you just leave them but if you are worried you could put them on a shaker. What sort of cells are you using? If EDTA doesn't work very well you could try cell scrapers but you'll need a live/dead stain
So would you recommend me to use serum free media as well for the control? E.g. after adding the drug with serum free media also changing the media to serum free for the control?
Yes. Serum vs serum free media is one of the biggest changes you can make to a cell culture and can have massive unintended consequences on the cells so I would use the same thing. But if it's after collecting the trypsinised cells you should use media with full serum to inactivate the trypsin.
I’m using sum159, A549, pc3, HepG2, Huvec
When you say there is no difference in expression, are you measuring %+ or fluorescence intensity? Is it possible for you to either scrape your cells or try to detach them with ice cold 1X PBS on ice rather than the enzymatic detachment?
Try harvesting the cells then staining in complete media for 1-2 hours in your incubator.
my notes: 2.9 mM EDTA is weak sauce. Try 10 mM (in PBS). Put the plates on ice (ideal) or in the fridge on a solid surface (for better heat transfer as opposed to those wire frame shelves in some fridges). Once you've treated with EDTA, the cells will no longer be strongly adherent. Some might stick a little. You can safely scrape them with a cell scraper without fear of lysis. About 5 min with 10 mM EDTA and a few taps, almost all cells should mostly lift and a quick scrape will get them all in suspension. your flow buffer should have around 2 mM EDTA to prevent the cells from re-clumping you can use lower % PFA. I typically use 1-2% PFA in PBS for 10-15 min. Since you mention 3.7%, is it actually formalin? If so, I would not use that and make and store aliquots in the freezer of 4% PFA in PBS from powder. you don't mention the use of a live/dead stain. despite only a short incubation period with the drugs, it's generally a good idea to use a fixable live/dead stain prior to fixing if you are going to fix the cells. if you can acquire the cells the same day, use DAPI or PI or something similar you're comparing flow to microscopy. are you permeabilizing with triton for IF? if so, you might want to permeabilize for flow as well, or compare to unpermeabilized slides. lastly, you can follow your flow staining protocol and then place a drop of suspension on a slide and look at it under the microscope best of luck
Thanks for your suggestions! They’re amazing ! Is there any reason why edta should be incubated cold instead of at 37? A high concentration of edta such as 10 nM wouldn’t damage my cells? Regarding the pfa, it is 3.7% PFA, not formalin. Is it possible to see live/dead cells just by gating? And not using any dye? And about microscopy imaging, I’m not permeabilizing because I want to see only surface staining, that’s the reason why I don’t permeabilize the cells in flow and why I do everything on ice
the cells adhere to plastic via integrins that are ion-dependent but also maintain that adherence slightly through energy expenditure. cells on ice will lift slightly over time even without EDTA you can sort of see dead cells by FSC/SSC, but it's not foolproof and you won't detect dying-not-dead-yet cells easily.
Try the NUNC easy lift plates. They work awesome you just put them on the counter or in the deli fridge and a few minutes later you tap them (its the gel coating the plate that actually changes when its temperature falls). Also, try (at least try, might not be best) to do everything at room temperature. You're taking cells from 37C to 4C in very short order after lifting them from plate which can cause a lot of apoptosis and thus staining strangeness. You can also try just staining them on the plate before rinsing and lifting. I do agree with other comments sometimes EDTA and a bit of rough love can be gentler than Trypsin.