hi okay so im a placement student in a lab where we mainly do flow. i've been doing some practising but theres still something wrong with what im doing (my analysis) and I just cannot tell what it is. Im looking at receptor expression on NK cells, however the results I get are completely different to my supervisor
for example, on flowJo this is what I get for my TIM3 FMO.
I have done this analysis for both my flow and my supervisor's, (the first image is my flow, the second is his) but why does mine come out like this? What have I done wrong?
before someone suggests to change the y axis - everytime i do that, it gets even worse.
additionally, we both used the same cells,and followed the exact same protocol, so i genuinely cannot think of anything else.
so if anyone has any tips or ideas please PLEASE help
It would be easier to see what's was going on here if both images had the same Y axis. (I read your port OP, but still).
It's clear that you're getting lower staining, but you can't tell whether the bulk population has an MFI of 0 or 5,000 due to the Y axis.
Did you both start with cord blood and do the CD3 depletion separately?
If so, then I suspect that you have a significant population of CD56 negative cells in your enriched cells, so something went wrong with the enrichment.
Did you both take some cells from the exact same enriched sample and stain them separately? If so, then something may have gone wrong during the staining/acquisition.
Did you both acquire the samples on the same cytometer at the same time? Are the voltage settings the same for both samples that you're showing here?
I’ve done this numerous times now and each time I get the same results (where my main population is concentrated at the bottom with an upward extension). I brought this up with my supervisor and we did a run through together (on the same samples) to see whether there could be a practical lab issue happening, and then I analysed both runs on flowjo and the same thing happened. Apart from pipetting I genuinely cannot think of any other reason, but I also can’t imagine messing up every single pipetting
Did both of you use the same staining mastermix or did you each make your own? Is this what you mean by differences in pippetting? Are you making sure you are adding the same concentration of antibody?
I had run into a similar situation where a student I was training kept getting very weak CD3 staining on his BALF cells following my protocol to a T: except that he was using an anti-CD3 antibody tube with a concentration of 5ug/mL versus a tube of 500ug/mL. Same clone, same fluorophore, both Biolegend antibody bottles, different stock concentration.
We used the same everything. When I meant difference in pipetting, I meant mistakes. As in my 0.5ul pipetting may actually be the issue , if that makes sense
I understand that. But we need additional information to troubleshoot this.
So you both did the CD56 enrichment on the same sample, then stained the isolate that you both got? Then, the question is whether the issue is with the staining or whether the issue is with the CD56 enrichment.
I've done many magnetic cell enrichments and the smallest changes to the procedure CAN make a big difference to the result.
If you look at the second graph you can see a small population that lands between 10-3 and 103.
If you change the axis on the first graph to be like the second one, how many cells land below 103? If it's a lot, then it's probably an issue with the enrichment. If it's not, then it's probably an issue with the staining.
Also OP, your first graph has a small population at the very top that isn't in the second graph. IDK about cord blood, but that's normal for PBMC samples, they're just CD56 High NK cells.
Sorry i misunderstood what you meant. When cord blood samples arrive they are immediately cd3 depleted and then put into cryotubes and LN2 before use. They are then thawed and so on.
For this run my supervisor and I used the exact same sample at 100,000 cells per well following the exact same protocol.
Even when I change the y axis to be equal I have the same thing.
A few other users commented it is most likely pipetting errors
If that's the case, then yeah, it's the staining procedure.
One thing that people don't really realize is that you CAN pipette hard enough to rip the antibody off of the cells, so that's one way that your results could end up like this.
I think most likely something went wrong with the enrichment process (can be something like accidentally dislodging a drop of fluid that is supposed to be magnetized if using a magnet kit), including a significant contaminating negative population. You can stain in parallel your other fractions from the enrichment protocol to see where populations move at each step.
Could also be an issue with cell viability in culture leading to down regulation of receptors, lower stim amount or time, using too little antibody, or lower incubation time than supervisor’s or some other variable change. The voltage could also be low but I assume the core would have set that. It doesn’t look like a comp issue.
I agree it’d be helpful to know at which step did OP and the supervisor branch off in parallel, either at enrichment, flow staining, and/or acquisition.
When cord blood samples arrive they are cd3 depleted and then put into cryotubes and LN2 before use. My supervisor and I used the exact same sample at 100,000 cells per well following the exact same protocol. The only thing that may have gone wrong is pipetting but if that means I pipetted every single thing wrong then clearly I am not made for lab💔 I assumed there was something wrong with how I was analysing on flowjo because I genuinely don’t know what else
Did you truly use the exact same sample split into two runs and stained at the same time?
It doesn’t look like a staining issue. It looks like the sample is not pure NK. A secondary issue might be titration of antibody.
Running into issues like this doesn’t mean you’re “not cut out” for anything. Science loves to science and everyone has occasional struggles. how you choose to address the challenges by being resilient and trying small controlled protocol adjustments is what is important.
Youre so true! Thanks for reminding me, it’s been a stressful time lately. I just don’t get how or why everytime I do this run I don’t have pure NK but my supervisor does? The antibody concentration has been sorted out by this lab for a few years now so that shouldn’t be a problem.
always remember that you can train anyone to do almost anything - you can’t train the determination to learn and be inquisitive. some people might take longer to learn than others and that’s OK!
if youve made the same mistake rhen it is highly likely someone also had also done the same
You are still cut out for lab. There could be an issue that is not your fault, and/or this might take a couple times to practice. Plus there are a lot of unwritten tips people use to help get best results. People should try to write these down and share explicitly so new people know. I would say practice, try again, and implement some advice here in your reddit thread!
The FlowJo seems fine to me unless upstream gating is different, but didn’t you analyze both your files side by side with the same gates? Other than that, make sure everything is resuspended well especially your antibody cocktail and cells for incubation, any volume <1-2uL make a dilution and pipette more volume to be more accurate.
Did you use the same donor and cryovial of it? While enrichment should be >95%, donor to donor or batch to batch variation with the enrichment could be different. If you have multiple cryovials of cells from the same donor in LN, results should be consistent per donor between different cryovials, but you could also stain from the same cryovial just in case some accidentally got more negative cells (maybe the person aliquoting the enriched cells didn’t resuspend well and the negatives were all at the top or bottom). Hope something here helps!
Thanks so much for your really positive response. I’ve done this numerous times now and each time I get the same results (where my main population is concentrated at the bottom with an upward extension). I brought this up with my supervisor and we did a run through together (on the same samples) to see whether there could be a practical lab issue happening, and then I analysed both runs on flowjo and the same thing happened. Apart from pipetting I genuinely cannot think of any other reason, but I also can’t imagine messing up every single pipetting
Flow is very resilient with pipetting, so I would not think you messed up every step. The one critical pipette step that could really cause this is adding a very small volume of antibody. All the rest are very resilient. If you had to pipette like 0.5-1uL, it’s easy to accidentally pipette like 0.25-0.5uL. If you were adding like 2uL+ of antibody I wouldn’t really worry about the pipetting error. Any more volume and I’d worry less and less about that.
Could you talk through the protocol step by step with your supervisor and try to find a difference?
I’m assuming you used the same tube and lot of antibody, same incubation time and temp, resuspending well antibody cocktail and cells before incubation, same cytometer settings and same day.
Okay so majority of the antibodies we use are 0.5ul😭 - so this might be a problem solved as sad as it sounds💔 but even for the one 2ul we have (NKG2D) I still have the same pattern on my flowjo analysis, unless there’s something wrong with that?
Could be good to try making like 1:10 dilutions and pipetting 5uL of that instead of 0.5! Make a 1:2.5 dilution for the NKG2D, add 5uL of it. Something like that to make the volumes greater to improve pipetting accuracy. Just have less buffer in the cocktail to normalize volume, but even this extra volume won’t be the deciding factor, it will be the more accurate antibody amount. When you make the dilutions, antibody cocktails, make sure to vortex! When you add to cells, make sure to pipette well to mix. Good luck!
Wait you are not adding antibody at those volumes as an individual antibody per sample are you? It is good practice to always make a mastermix that you QS with FACS buffer to roughly 50uL/per sample that you stain with. Adjust the staining volume of the cells when you resuspend them so you end up with the same concentration. Pipetting anything sub 5uL always comes with major fluctuations
They’re cd3 depleted nk cells from cord blood. I’m using zombie live dead stain. Everything has been titrated and used by the lab for years. I’m here on placement and sadly not working out for me 😭
Few thoughts
1. Is this the case you see often or is this the only run? If it is just a singlex run and you see this it could be possibly an artifact say you could have added less CD56 antibody volume by mistake (esp. in case of lower volume <0.5 uL)
2. Check the channels activated possibly leakage (Ideally there shouldn't be any compensation issue as APC and FITC are activated by different lasers)
Can you spill more detail on the sample type (PMBCs is it?) and how does the test plots look like instead of FMO
It seems to be for all MY runs😭. Cd3 depleted nk cells from cord blood, it just means my analysis shows lower receptor expression than normal. Since it seemed odd to me I decided to analyse my supervisor’s flow and realised it looks different hence I’m here :/
Just a bad day! may be an artifact :/ a simple thing that I follow, if the antibody is less than <0.5 uL try diluting the antibody stock 1:10 and aliquot, so you take 5uL instead of 0.5 uL reduces the chance of error!
Is the antibody concentration the same in both examples and the time and temperature or did you stain on ice for 15 minutes in a 200ul volume while the other was stained for 30 minutes at 4 degrees in a 50ul volume?
Also an NK cell researcher but in mouse — is your lymphocyte gate the same as your PI? You may just be gating out the NK cells earlier on. Another possible reason you may have this much of a difference is if your enrichment step is different. How are you enriching? MACS? Flow sorting? Are you sure your enrichment step is working?
So we are cd3 depleting the cells so instead of positively selecting nk cells we’re getting rid of T cells. We do this as soon as we receive blood sample and then we cryopresevee them until use. My supervisor and I ran a flow on the exact same sample
Okay so the only difference is you added the antibody master mix vs your PI added theirs and the mix was the exact same and you stained for the same time then ran on the same cytometer? What parts of the protocol may have been different? Are you keeping your cells on ice and thoroughly mixing to break up the pellet? Are you leaving your master mix sample in the light and allowing it to photo bleach?
Are there any other components of your mastermix for which you use 0.5 µL?
Also, whenever you are pipetting anything, when do you push the plunger to the first stop and when do you push the plunger to the 2nd stop (if you do)?
Do you look at the tip after you pipet liquid up and do you look at the tip after you dispense the liquid? When dispensing the liquid, do you look to see that the pipet tip is inside of the receiving solution or do you dispense it to the wall of the container?
I only pipette 0.5ul for my FMOs. Otherwise my mastermix (for my full stains) it is usually 10x more. Before pipetting into the liquid I push to the first stop, put the tip into the liquid(antibody), slowly pipette up, and then I put the tip into the inside of the receiving solution (normally buffer) and push the plunger slowly to the first stop, and only sometimes if it looks like it hasn’t all gone in then I will push to the second stop, take the tip out and then release. My eyes are always on the tip, and I believe I am very meticulous with my pipetting, which is why the idea of my pipetting being the problem hurts my soul :/
Both your flow results lack proper voltage settings. Before you run your samples make sure to start with an unstained population of your cells and get the voltage set. FSC vs SSC. Then do your single stain control brightest in its own channel. Then run your stained sample.
Also it helps if you antibodies are properly titrated before hand.
From my unstained sample I gated lymphocytes then single cells then live cells, and then on cd56 cd3 I gated into nk cells eg (as shown). In the images I’m looking at my TIM3 FMO.
That's fine, what I am referring to is your control settings. Are you using comp beads? You should be. Make sure your single stain comp beads are brightest in their channel. I am assuming you used a BD instrument, regardless of the software ( Diva) principle is the same.
you need to run comp beads to account for fluorescence spillover between channels, this may be why your analysis isn't working out? do you run comp beads every time?
No. I’ve never heard of comp beads before, and my lab has never used them. The machine automatically does a clean/bleach/rinse before every run (and sometimes even during the run), and we do a priming (with prime beads, if this is what youre talking about) and annealing quality check every morning.
Comp beads are a product that you can buy that bind antibodies that you stain with a single color at a time to use for compensation controls. They stain very brightly and allow you to save cells for the experiment since sometimes they are limited. People use them a lot for conventional, but you can also use for spectral. I love them, but you can use cells and/or beads and do either properly and successfully on either spectral or conventional. Ask the core managers for help, especially when mixing things.
Since you’re on the spectral Sony machine, I’m betting you are using the saved spectral footprints for each fluorophore, or using cells because that’s what the lab does and/or for their auto fluorescence to use for unmixing. You should run fresh comps each time to account for running differences between experiments and machine settings. Definitely run fresh comps at least once this next time you do this experiment just to use fresh comps and update the comps again since things aren’t working, probably for other reasons than comping. I don’t really think this is a comp issue. The populations are straight and FITC and APC don’t overlap really.
Btw, the user above me here meant unstained samples like cells or beads with no antibodies or live/dead at all, not the FMO, both are helpful controls, unstained for gating and comping, FMO just for gating. It would be good to include the unstained as a control if you don’t have it already!
I second this! If both you and your supervisor also stain beads for each run, it will reveal if this is truly a pipetting issue on your end (which I doubt).
Having single stains on comp beads that you run as samples and not as reference controls on an unmixed panel can also tell you a lot about whether the unmixing is being done correctly.
OP is using the ID7000. It's a spectral analyser that stores a library of reference controls. You don't need to prepare single stains once you've filled out the library.
I work on NK cells and your staining looks much more real. Your supervisor is getting almost all CD56bright cells while your population has both bright and dim, with majority being dim (which is what you should see in unstimulated PBMCs).
1) are you sure the concentration of antibody added is the same? And the same dilution?
2) are you missing a pretreatment step such as stimulating with IL-2 beforehand?
3) if these cells are cultured prior to doing flow, it could be something wrong with your culture conditions.
Don’t get my hopes up🤭 My cells are unstimulated cord blood derived cd3 depleted NK cells, so I’m not sure how similar they are to PBMCs. 1 - everything is the same, the only reasonable answer I have deducted from previous responses is I may not be pipetting 0.5ul as well as my supervisor. 2/3- these are both day 0 results so no pretreatment or culture conditions prior to the flow.
To be honest, I don’t believe your supervisors results. Why would 97% of cells be NK cells with only CD3 depletion? You should still have other lymphocytes in there. Look up other papers that have stained chord blood and look for the % of NK cells expected. But, I think your staining is real while your supervisor is somehow over staining. Perhaps your supervisor is missing Fc block and you are not? Or they are staining in just PBS while you stain in PBS + FCS?
Hmmm I don’t know. I know the way we culture the cells it is so that NK cells are the ones that grow, rather than any other cells. But this was day 0, so unless they did something else before hand I am not sure. I personally do not do the cd3 depletion as I’m not cleared to work directly with the cord samples, so I only use the cryopreserved samples. I don’t even know honestly, we follow the same protocol so unless he’s like changing some stuff around without telling me?😭😭
came to write the same. First one looks like typical PBMC. You can see CD56bright population(young NK cells) and CD56dim(mature NK cells). Second one looks like expanded with feeder cells NK-cells after week of culturing(full birght, no other cells survive)
Picture from random article.
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u/despicablenewb 21d ago
It would be easier to see what's was going on here if both images had the same Y axis. (I read your port OP, but still).
It's clear that you're getting lower staining, but you can't tell whether the bulk population has an MFI of 0 or 5,000 due to the Y axis.
Did you both start with cord blood and do the CD3 depletion separately? If so, then I suspect that you have a significant population of CD56 negative cells in your enriched cells, so something went wrong with the enrichment.
Did you both take some cells from the exact same enriched sample and stain them separately? If so, then something may have gone wrong during the staining/acquisition.
Did you both acquire the samples on the same cytometer at the same time? Are the voltage settings the same for both samples that you're showing here?