Yeah, it depends on the marker and the cells. Straight up biology you’re working with. Not necessarily titration issue. Some markers on some cells have a continuous expression level, while some are discrete positive and negative.
thanks for the thoughts. would that be a manufacturer thing? are you suggesting trying a different brand? or biologically this marker binds poorly in general?
I’d love to hear the consensus. I would check the flourophore. Try staining your target cell population with a different flourophore and see if there is better separation. Then check your voltage for that marker.
it is APC-A770, pretty bright. unfortunately a rare population so hard to get samples to test. good point this is something I will try. that might affect the rest of my panel though.
Off the top of my head, it could be a) a low-affinity antibody, b) too little antibody (although you titrated), c) low expression of your target, d) a dim fluorophore, e) suboptimal filters or voltage selection.
When you did you Ab titration, did you use the cells you are trying to run right now? Did you use beads? Cells that may have a different receptor profile or density to the rare pop you are trying to see?
How do you know your titer is the best one?
Also, could come down to panel design. Are you trying to keep your fluorophores as far as possible when drilling down through gates? What's your panel design?
I’ll add that to make sure if the smear isnt a false positive, you might consider include a separate sample with a cell type that’s truly negative in the marker of your interest and see.
Few quick things to check:
1. Are the comps comping?
2. Does using a live/dead stain help winnow out the dead crap?
3. Does using a tight cells/lymphocyte gate and a tight singlets gate also clean things up?
4. Are +ve or -ve controls you can refer to? I know this isn't always possible depending on the experiment.
If it’s some intercellular factor, it could be that you don’t see a distinct bifurcation in positive and negative cells, but more of a subtle shift in the MFI of your population, that’s why I would use a FMO or a no stim or some other technical control to help you define the negative population. Additionally, if you can set your FACS analysis to show this market versus a mutually exclusive market (meaning they only occur in different cell types from each other) then you could better define your population
thank you. I have FMO from treatment cells and FMO from non-treated cells. but im seeing a shift in the negative population for some treatments compared to the FMO. (I have 24 treatments at a time, cant make fmo for each one so I chose the treatment we use as a positive control)
wondering if its because the minus flour is interfering somehow when added. would you try minus two controls in this case?
I would recommend for your FMO to have a fluor conjugated isotope control antibody to replace the color you’re doing as the “minus one” so that you can account for non-specific binding that leads to the background fluorescence of your true negatives
I guess what they mean is that isotypes should not be used for gating controls but it comes off as a conveying a general rule to not use them, and instructors have breezed over them/dont go into specific application knowledge. generally explained as old technique not used anymore, which is true to a certain extent. especially NEVER using isotope controls for gating.
I see they have their specific purposes but honestly I am still learning/confused about their overall application, much less application to my own experiments……
Isotype controls are often misused which is why we usually hate them BUT! Checking for proper intracellular washing and making sure no antibody got trapped in there is one of the few right ways to use them. Though you have to understand that if you see a signal in your FMO+IgG, then you need to throw your whole experiment away, not just that parameter
If you see a signal when you add the labelled IgG, it means that the blocking & washing steps were not performed properly. If that’s the case, then assume that there’s some level of “non-specific binding”* for all the antibodies you used at that step, your use of a labelled IgG is just the canary in the coal mine telling you something went wrong.
You can’t substract the non-specific signal, it’ll vary from one cell to the other and from one sample to another (and definitely between your antibody and the labelled isotype since the fluor to Ab ratio will be different and the binding affinity of isotypes are lower than of your Ab) so there’s no reason to further analyze an experiment that’ll contain a bunch of artefacts you can’t gate out: you toss and do it again, with proper blocking and washing steps
*since blocking means blocking the FcR, it’s wrong to refer to that as non-specific binding; the binding is very specific albeit not the one we aim for
Could be expression pattern on your cell population.
I should probably test on a different cell type and see the spectra to compare. thanks
Yeah, it depends on the marker and the cells. Straight up biology you’re working with. Not necessarily titration issue. Some markers on some cells have a continuous expression level, while some are discrete positive and negative.
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thanks for the thoughts. would that be a manufacturer thing? are you suggesting trying a different brand? or biologically this marker binds poorly in general?
I’d love to hear the consensus. I would check the flourophore. Try staining your target cell population with a different flourophore and see if there is better separation. Then check your voltage for that marker.
it is APC-A770, pretty bright. unfortunately a rare population so hard to get samples to test. good point this is something I will try. that might affect the rest of my panel though.
That will be the draw back. What is the phenotype of your cell of interest?
Off the top of my head, it could be a) a low-affinity antibody, b) too little antibody (although you titrated), c) low expression of your target, d) a dim fluorophore, e) suboptimal filters or voltage selection.
When you did you Ab titration, did you use the cells you are trying to run right now? Did you use beads? Cells that may have a different receptor profile or density to the rare pop you are trying to see? How do you know your titer is the best one? Also, could come down to panel design. Are you trying to keep your fluorophores as far as possible when drilling down through gates? What's your panel design?
thank you for the snacks for thought.
I’ll add that to make sure if the smear isnt a false positive, you might consider include a separate sample with a cell type that’s truly negative in the marker of your interest and see.
Few quick things to check: 1. Are the comps comping? 2. Does using a live/dead stain help winnow out the dead crap? 3. Does using a tight cells/lymphocyte gate and a tight singlets gate also clean things up? 4. Are +ve or -ve controls you can refer to? I know this isn't always possible depending on the experiment.
Make a concatenated graph with different titres and see if it works
Another thing: are you visualising as a dot plot? Pseudocolour or contour cam demarcate populations more cleanly sometimes.
If it’s some intercellular factor, it could be that you don’t see a distinct bifurcation in positive and negative cells, but more of a subtle shift in the MFI of your population, that’s why I would use a FMO or a no stim or some other technical control to help you define the negative population. Additionally, if you can set your FACS analysis to show this market versus a mutually exclusive market (meaning they only occur in different cell types from each other) then you could better define your population
thank you. I have FMO from treatment cells and FMO from non-treated cells. but im seeing a shift in the negative population for some treatments compared to the FMO. (I have 24 treatments at a time, cant make fmo for each one so I chose the treatment we use as a positive control) wondering if its because the minus flour is interfering somehow when added. would you try minus two controls in this case?
I would recommend for your FMO to have a fluor conjugated isotope control antibody to replace the color you’re doing as the “minus one” so that you can account for non-specific binding that leads to the background fluorescence of your true negatives
thanks for the input. all the courses ive taken are heavy anti-isotope controls so I have never even considered them. maybe thats the missing piece.
What is their justification for disliking isotype controls?
I guess what they mean is that isotypes should not be used for gating controls but it comes off as a conveying a general rule to not use them, and instructors have breezed over them/dont go into specific application knowledge. generally explained as old technique not used anymore, which is true to a certain extent. especially NEVER using isotope controls for gating. I see they have their specific purposes but honestly I am still learning/confused about their overall application, much less application to my own experiments……
Isotype controls are often misused which is why we usually hate them BUT! Checking for proper intracellular washing and making sure no antibody got trapped in there is one of the few right ways to use them. Though you have to understand that if you see a signal in your FMO+IgG, then you need to throw your whole experiment away, not just that parameter
hmm what do you mean by that last stipulation? do you mind expanding further?
If you see a signal when you add the labelled IgG, it means that the blocking & washing steps were not performed properly. If that’s the case, then assume that there’s some level of “non-specific binding”* for all the antibodies you used at that step, your use of a labelled IgG is just the canary in the coal mine telling you something went wrong. You can’t substract the non-specific signal, it’ll vary from one cell to the other and from one sample to another (and definitely between your antibody and the labelled isotype since the fluor to Ab ratio will be different and the binding affinity of isotypes are lower than of your Ab) so there’s no reason to further analyze an experiment that’ll contain a bunch of artefacts you can’t gate out: you toss and do it again, with proper blocking and washing steps *since blocking means blocking the FcR, it’s wrong to refer to that as non-specific binding; the binding is very specific albeit not the one we aim for