Hi , I would like to have these subject as PowerPoint slide if it’s possible
@user-rg2zn8xx6jАй бұрын
Hi, can I have the PowerPoint for these videos please?
@fatimainigo25Ай бұрын
Great tutorial! Thanks a lot!
@eduardoduarte19683 ай бұрын
Thank you for sharing this. I learned a lot!
@byounghaan52495 ай бұрын
Thank you so much~!! It is really helpful.
@JinhyunPark-ws6sr7 ай бұрын
thank you for the informative video!
@john121cena8 ай бұрын
thank you so much for your help and this amazing work
@mervet6438 ай бұрын
Also I will add your Work about Flow cytometry after 6 month with my thesis from Turkiye republic.
@paulchang87439 ай бұрын
Can this approach substract the autofluorescence?
@User9527_10 ай бұрын
Thank you for the tutorial! Very helpful. One more question: you stained 1M cells in 100ul buffer flow analysis with around 0.5-1ul antibody. For sorting, how much volume buffer and how much antibody do you use? Could you please give a suggestion? Thanks!
@paporisharma2936 Жыл бұрын
Excellent video, Thanks
@biscotrash Жыл бұрын
Hi, Awesome video. How are you preparing the dilution tubes though? Are you diluting them serially, or spiking in the precise number of ug to each tube? I'm asking because i'm doing a titration for the first time for a large panel, so many tubes, and i'm trying to figure out the easiest way to set up the tubes. Thanks!
@UChicagoFlow11 ай бұрын
Hi @biscotrash - serial dilution is the way to go, it makes it much easier to handle the lower concentrations of your standard curve!
@DavalgaanHailtuud Жыл бұрын
Nice
@kellyyunqianwei5882 Жыл бұрын
Thank you so much for the video! much better training!
@hedyxu7184 Жыл бұрын
very helpful for a new user
@arora1991 Жыл бұрын
So grateful for this video! thanks a lot :)
@florianmarquet4324 Жыл бұрын
series of high quality presentations, I have been doing cytometry for 15 years, this will allow me to explain with precision what I was doing without sometimes knowing why
@hanaabdullah66622 жыл бұрын
Thank you for these useful videos, is there a way to contact you by email? Thanks
@UChicagoFlow2 жыл бұрын
Hi Hana, found a new position in industry and no longer works at the UoC. We'll be happy to help - find us on google and follow the contact us link.
@hanaabdullah66622 жыл бұрын
@@UChicagoFlow Thank you so much for replying. I tried but I was not able to find you.
Hi, we are getting error while running fluidic startup..after few minutes of run it's showing a error to fluidic startup has been shutdown due to error. Can you help me out to find the solution
@UChicagoFlow2 жыл бұрын
Hi! I'm guessing this happening on a Canto? I haven't seen one of these in years, by my guess would be an issue with the sheath pressure not getting high enough to go through startup. I'd check the pressure gauge on the fluidic cart, purge any air stuck in the fluidic line, your tubing connectors for any leaks. Again, wild guess here! Can't be sure without having a look at the instrument.
@mikhaeldito2 жыл бұрын
Very neat trick!
@BrendaRaud2 жыл бұрын
Thanks for the video! It really is a great resource for those trying to learn how to use the Aurora cytometers. In the case we acquire everything and later on (the next day) we realise that our reference samples were not optimal, what is the best way to re-acquire them? is it ok to just open the experiment the following day after running the QC, replace the references (i.e. overwrite them) and do the unmixing again?
@UChicagoFlow2 жыл бұрын
You could overwrite the references. Or it may be better to add new tubes in the reference group so that you have all the options when unmixing the data. It's easiest to do this on the actual aurora - if you ever choose to do unmixing on another computer you'll have to move some instrument settings as well. Depending on how complex your experiment is, you might find it helpful to discuss with your Cytek Technical Applications Specialist to advise you on the best approach for your specific experiment.
@pavlabohacova59062 жыл бұрын
@@UChicagoFlow Thank you for the stunning presentations! Please, how to transfer instruments settings between computers? I'm planning to unmix/analyze data on another computer to save machine time. Thanks!
@UChicagoFlow2 жыл бұрын
@@pavlabohacova5906 You'll need administrative rights to make this change, so it may be best to talk to whoever maintains your Aurora. You'll need to transfer instrument settings using the data maintenance tool. Also my understanding is that you only need to do this if you're unmixing files that were recorded on different days. If everything was run on the same day you just need the fcs files/experiment.
@feitu64032 жыл бұрын
It is a great series to introduce Flow cytometry. Here is a question for FMO and unstaining control. Could I use beads without antibodies for unstaining control or i have to use cells without antibodies for that? The same concern for the FMO, cells or beads? Thanks.
@UChicagoFlow2 жыл бұрын
Hi Fei, thanks for the good word. Unstained beads will be used as control for compensation and unmixing if you are using the same beads for your positive group. The autofluorescence of both positive and negative must be the same. But for your FMOs, the question is different: you are trying to figure out the impact of all of your fluorophores (minus the one you're gating on) on the spread of the negative population. So the only appropriate way to create this control is to use cells.
@feitu64032 жыл бұрын
@@UChicagoFlow thanks for your response. I am still confused with the unstained control. In my previous experience, I used unstained beads to adjust FSC and SSC to settle the cell population in the middle of plot. And set up the voltage of each color through the single control with beads for compensation. However, I didn’t do unstained cells to adjust that cell population. Beads could not the same size with the real cells. So, I want to know whether my design is acceptable through unstained beads to gate cell population instead of unstained cells? Sorry for my tedious words.
@zxcv26772 жыл бұрын
@@feitu6403 I typically set voltages on FSC and SSC specifically for the beads and cells. Most of the time, they have different properties, and will require you to decide on the settings to best showcase what you're actually looking at. As far as selecting the voltages for the fluorescent parameters detectors, I think your method likely puts you within range of what will allow you to separate your positive and negative markers without saturating the detectors. This is the whole point and if that is what you have right now, you are probably good to go. We go in more details about voltage selection in the next video in this series - here 13:05.
@feitu64032 жыл бұрын
@@zxcv2677 thanks a lot!
@smita88ful2 жыл бұрын
How to run both cells and beads of same colour in one experiment? please suggest!
@UChicagoFlow2 жыл бұрын
When creating or editing your experiment, on the group tab select create or edit reference group, then in the window that lists all of the reference control tubes there is an "Add" button in the lower left corner that will allow you to add as many reference controls as you need.
@timothyborgogna72092 жыл бұрын
I hope your department recognizes you for how much work you put into these. Thank you so much!
@annalau96452 жыл бұрын
Bravo!!!!
@clauzugomvz3 жыл бұрын
How can I get to the link at 14:51?
@UChicagoFlow3 жыл бұрын
Hi Claudia - Good catch, we'll figure out a solution on accessing the link on the slide. It's on the CAT Facility Blog: voices.uchicago.edu/ucflow/2020/01/16/how-many-cells-should-i-stain-the-impact-of-cell-concentration-and-antibody-concentration/
@kyshark13 жыл бұрын
Awesome explanation. Very useful and many thanks for posting these videos 👍
@ObsoleteTutorials3 жыл бұрын
Can you mix the beads and cells for the reference controls and stain them in the same tube? Then tell the software which populations to use as positive and negative populations
@UChicagoFlow3 жыл бұрын
I would not recommend combining the beads and cells into a single tube two two reasons. First you would need to be 100% certain that the beads and cells do not overlap on the FSC vs SSC plot. Depending on the tissue used, there could be some overlap. Inability to separate beads and cells could be problematic for how autofluorescence is accounted for in terms of calculating unmixing from the reference controls. Second is that beads and cells don't necessarily require the same concentration of antibody for optimal results. I could come up with a bunch of advanced tricks that would allow you to combine beads and cells into the same tube, but ultimately it's going to take a lot more effort than it's worth.
@Teresa-xt8oi3 жыл бұрын
Thank you for the great tutorial! For Unmixing of stimulated PBMCs, do you suggest to include viability dye to avoid unspecific Ab binding to dead cells? And is it then also possible to do unmixing with the tool box like in the video? By including a single stained viability dye tube? Would really appreciate your advice! Thank you!
@UChicagoFlow3 жыл бұрын
Personally, I have not found that including a viability stain for titrations was necessary. If you do have a high percentage of dead cells then you may find it beneficial to include a viability stain. I don't think people typically add in the viability stain with titrations unless they're struggling to interpret the data without it. The extra tools menu will let you unmix any number of fluorophores, just like the unmixing wizard in the acquisition tab.
@Teresa-xt8oi3 жыл бұрын
@@UChicagoFlow Thank you very much for your expertise! Really highly appreciated! If I dare to ask, I would have 1 question concerning wet lab work. For the final staining, we would use 1 x 10^6 cells in 100 µl. For the titration, we would like to use less cells, eg. 100.000. Would you suggest to keep the volume constant (use 100.000 in 100 µl) or would you suggest to use less volume. Of course we would keep the final antibody concentration constant. I would really appreciate your help, since the literature on this topic is not quite clear!
@chugcytometry32843 жыл бұрын
@@Teresa-xt8oi There is a blogpost on the CAT Facility website with data supporting the idea of keeping the volume the same for your controls : voices.uchicago.edu/ucflow/2020/01/16/how-many-cells-should-i-stain-the-impact-of-cell-concentration-and-antibody-concentration/
@Teresa-xt8oi3 жыл бұрын
@@chugcytometry3284 Thank you for your help!!
@cmontenegro88163 жыл бұрын
Thank you for your video.I’m analyzing human cultured adipocyte tissue derived cells. These cells are already cultured and passaged several times. To obtain a single cell suspension is it necessary to pass the cell suspension through a cell strainer? I’m worried because of the diameter of the ASCs I might lose the cells
@UChicagoFlow3 жыл бұрын
I'm sorry, I'm not familiar with this cell type so I don't think I can advise you. I don't think cell strainers are an absolute requirement, but they work well for cells that are often used in flow cytometry. You'll have to find a method of single cell preparation that is appropriate for your cell type.
@cmontenegro88163 жыл бұрын
@@UChicagoFlow Thank you for taking your time to reply. I appreciate it. I will continue to search for the optimal conditions.
@zoyageorgieva66843 жыл бұрын
Is it possible to adjust the spillover matrix in another programme (e.g. FCS Express post-unmixing; or FlowJo if you have unmixed the files already and re-imported the unmixed files into flowjo?). So far I am finding I can't apply compensation in either of these programme to tweak the unmixing results (but I am pretty new to this).
@UChicagoFlow3 жыл бұрын
Yes, it is possible. Just to make sure we're all on the same page: unmixing can be done in multiple applications (SpectroFlo, FlowJo, FCS express), though I personally find SpectroFlo the most user friendly. With SpectroFlo unmixing there is no way to view the "unmixing matrix". If you want to make a manual adjustment, you must use a separate method - compensation. Compensation can be done in multiple applications (SpectroFlo, FlowJo, FCS express) so that compensation is essentially applied on top of unmixed files. I would recommend manual compensation, not an automated compensation wizard. I am less familiar with FCS Express, but in FlowJo you must open the compensation editor and click "edit", which will generate and apply a new compensation matrix. To get a negative comp value you can select a box in the matrix and hit the down arrow key - typing a negative sign doesn't work but once you have a negative value you can manually type any value after the negative sign. And I feel the need to reiterate the most important point: manual compensation should be used with caution and for data that will be presented/published you are only allowed to adjust the compensation to correct the single stain controls and then apply that to the fully stained sample. Never ever adjust compensation directly on a fully stained sample.
@ziyadarahamto63353 жыл бұрын
Good video and Please can u have more information in flow
@UChicagoFlow3 жыл бұрын
Thank you very much! Which topic would you like us to cover next?
@junaidrehmani98293 жыл бұрын
@@UChicagoFlow Please upload a detailed video of system calibration , standardisation and laser delay.
@kaylaparr77323 жыл бұрын
Thank you for the tips on titrating using the Aurora. A bit of a naïve question but do you need to unmix since all samples are either unstained or single color? I'm wondering if I can export the FCS files after acquiring using the Default experiment and then just analyzing in FlowJo. Thank you again!
@chugcytometry32843 жыл бұрын
The overlooked point about the Spectral flow instruments is that you can, as you mention, just use the raw data without unmixing. You'd essentially be using the instrument as a traditional flow cytometer, and that is totally fine. The width of the bandpass of each detector is usually much smaller than what you'll find on you typical flow instrument, so fewer photons will hit the detector. But if the signal is bright enough, it should not matter.
@kaylaparr77323 жыл бұрын
@@chugcytometry3284 Thank you for the helpful response! I just titrated antibodies using mouse lung and was worried why a few populations hadn't resolved (CD206 BV605 and Siglec-F BV510). I'm hoping that it's a brightness issue that will be resolved once I go back and properly unmix the files this week.
@UChicagoFlow3 жыл бұрын
This question comes up a lot, and I don't think theres a consensus on which way is best. I think a lot of people end up trying both ways.
@chugcytometry32843 жыл бұрын
@@kaylaparr7732 Do you mean that you don't have a positive signal at all, or that you can separate your positive cells from the autofluorescence?
@kaylaparr77323 жыл бұрын
@@chugcytometry3284 Separating positive cells from autofluorescence. There is positive signal in all serially-diluted antibody samples but no clear positive and negative populations (all tubes, including unstained, look like a lot of fluorescence spread).
@vanessamoraes67493 жыл бұрын
Hey Laura! Thanks for sharing all this! Very helpful. I saw you make an overlay of unstained, fully stained, and single stained to make the panel quality check. When you overlay these, do you use all uncompensated parameters? (Maybe this question does make sense but I got confused when I was doing it as I have different histograms depend on my parameter on X axis). Thank you very much!
@UChicagoFlow3 жыл бұрын
If you just use spectral unmixing in SpectroFlo and do not use any compensation in addition to that, then the "uncompensated" and "compensated" parameters in FlowJo are the same. If you did apply compensation on top of the spectral unmixing (either in SpectroFlo or FlowJo) then you'll want to choose the compensated parameters in FlowJo. It's best to do a panel quality check in data that is free of any unmixing errors.
@emberthong75033 жыл бұрын
Thank you!!
@shokoufehkarimi64603 жыл бұрын
I love all your videos
@vanessamoraes67493 жыл бұрын
Amazing for me: a beginner on flow!!!
@patrickbier803 жыл бұрын
great tutorial and lol on the floppy disk ; - )
@gp43553 жыл бұрын
Thank you so much for creating these detailed guides. They have answered many small questions that used to bug me. I am so grateful your flow videos.
@UChicagoFlow3 жыл бұрын
You're very welcome!
@THE_LN_PLAYS3 жыл бұрын
Is it necessary to perform doublet discrimination and viability analysis when performing antibody titrations?
@UChicagoFlow3 жыл бұрын
Hi Lazar, Generally, I would say no. Most people titrate antibodies as a single stain. Doublet discrimination is important for ensuring that we're not getting false positives. Imagine if two single positive cells are stuck together - without doublet discrimination we might think we have a double positive cell. Because there's only one antibody to titrate in one tube the doublets aren't really a concern. Viability is also important for accuracy of your experimental data, your antibody might be nonspecifically binding to dead cells. See this blog for further explanation: voices.uchicago.edu/ucflow/2020/11/19/how-to-identify-problems-with-panel-design-bad-data-part-2/. For titrating an antibody the dead cells usually don't interfere too much with determining the optimal concentration. But I guess if you're seeing a much higher frequency of positive cells than expected or it seems like the background is really high maybe it's worth it to include a viability dye. I've yet to run into that situation, but I wouldn't say it's impossible. In that case you'd have to then worry about compensation between the antibody and viability dye, which is just more effort than a single stained tube. The simplest experiments are ideal.
@AngelaNess113 жыл бұрын
Great video! So detailed and informative! Thank you!!
@THE_LN_PLAYS3 жыл бұрын
Thank you for an excellent course. I am new to flow cytometry - how do you ensure that a single stained control / compensation control is brighter than overall sample, if the cell population we are targeting (in a compensation control tube) has low expression of surface marker? I would assume that a fluorophore-conjugated low expression surface marker would not emit high fluorescence signal due to small number of cells expressing said marker, even if the cell suspension is stained with a bright dye such as PE. Thank you for your help.
@UChicagoFlow3 жыл бұрын
Hi! This can be a tricky question. First I want to point out that the frequency and intensity of a marker are distinct features and independent from each other. You can have a low frequency population (<1% of the total cells) that expresses 100,000 molecules per cell (high expression/high intensity). Alternatively, you can have a high frequency population (maybe 60% of the total cells) that expresses 2,000 molecules per cell (low expression/low intensity). For a single stained, control the fluorophore intensity of the control must be as bright or brighter than the fully stained sample - regardless of the frequency of that positive population. If the positive population is rare, then you may need to record more total events in order to get enough positive cells, but this does not affect the intensity of the marker. If it is difficult to get enough positive events (100 would be the absolute minimum, but more is better), you may consider using compensation beads. My best recommendation is to stain the single stained controls and fully stained samples in the exact same way - tissue type, cell concentration, antibody concentration, incubation time, incubation temperature. The easiest way to mess up the controls is if people use one tissue type for their controls and another for their fully stained tube and it turns out that the tissues have different levels of expression for a marker. Or mistakes can be made in pipetting the single stained controls - especially when pipetting volumes less than 1 uL.
@prateekarora4143 жыл бұрын
Why we can not use CD4 BV750 (Tandem dye) as a reference control for unmixing the spectral signature of FoxP3 BV750 in the test sample?
@UChicagoFlow3 жыл бұрын
Each time a tandem dye is created, there may be slight variations in the signature. Meaning the signature from lot 1 may be slightly different from lot 10. Because the signature of the dye in the control must be the same as the signature in the sample, you must use the exact same vial of antibody to stain the control and the sample. If you use a different vial of antibody (such as a different lot or an entirely different antibody with the same fluorophore), you will likely not have the same signature in your control and sample and will end up with unmixing errors. Non-tandem dyes do not have this lot to lot variability and so you can switch antibodies or lots for those fluorophores.
@prateekarora4143 жыл бұрын
@@UChicagoFlow Yes, Indeed we can prepare the BV750 reference control from the same antibody lot that is going to be used for the sample. There is always a slight variation in the staining pattern/signature in tandem dyes from lot to lot due to fluorescence/protein ration, the pairing of tandem molecules/fluorophores etc.
@prateekarora4143 жыл бұрын
In Rule number 05, they are saying that all the controls must be treated with the same buffer as your sample but if the buffer changes the spectrum signature of reference (compensation) control then where would the problem occur when applying unmixing to the test sample? if we already set the spectral signature with the bright individual reference control then it shouldn't matter whether the test sample treated with permeabilization or fixation.
@UChicagoFlow3 жыл бұрын
Hi! Rule 5 states that the signature of the control must be the same as the signature of the sample. Fixative can slightly alter fluorophore signatures, so if your controls are unfixed and your samples are fixed the signatures will not be the same and you will get unmixing errors. You must either have both controls and samples fixed or both controls and samples unfixed.
@prateekarora4143 жыл бұрын
@@UChicagoFlow It means to say that the unmixing is different from conventional compensation where it says that the compensation control should be brighter than your sample control. So, for the unmixing algorithm/feature, we always need to use a similar treatment or conditions for references and samples to get proper unmixing results. As in the compensation rule, it says that the background fluorescence should be the same for negative control and positive control (Means if there is a treatment in your positive control/reference then the similar treatment should be given to negative control/reference, no comparison with the sample condition/treatment)
@UChicagoFlow3 жыл бұрын
@@prateekarora414 Actually, the rules to create proper single stain controls are the same - whether you decide to use them to perform spectral unmixing or compensation. Since compensation can be performed manually, people are often able to compensate their data even if their single stain controls do not follow all of the rules. And because conventional cytometers only look at a fraction of the overall emission spectra, they may not be able to detect lot-to-lot variability as well as spectral cytometers, so using a different lot of antibody between controls and samples may not have as big of an impact on compensating data as it does on spectral unmixing. Your comment also referred to a "compensation rule" - I'm going to assume you meant the third rule in my video. I'm not sure if you understood this rule correctly and what you mean by "treatment" (fixation? stimulation? disease model vs control?). It's challenging for me to provide detailed explanations in KZbin comments - if you have questions about what controls to use for your experiment, I would suggest contacting your Cytek Technical Application Scientist and they should be able to discuss this with you.
@prateekarora4143 жыл бұрын
@@UChicagoFlow Thanks and appreciate your valuable time to give such commendable comments. I do apologize if, my comments annoyed you. I completely understood the concern of rule 03, where it says that the autofluorescence of the negative population in the positive control should be identical to the autofluorescence of negative control (Highly recommended in spectral cytometers to matching the background for better unmixing) else use universal negative in third party software. I mean of treatment was for buffers only means as you said in rule 05 that all the controls should be treated with the same buffers as your sample to get the identical spectral signature but if someone is using beads then I presume there is no need of treating the beads with the same buffers as for the samples, I know the treatment will alter the fluorescence spectrum but here if we add the treatment buffer like a fixative buffer to the beads references controls then the spectral signature would not be similar to the cells treated with same fixative buffer..
@UChicagoFlow3 жыл бұрын
@@prateekarora414 I'm sorry if I sounded annoyed, that was not my intention! I just want to make sure you're getting the answers to all of your questions and I am not able to answer everything in full detail using KZbin comments. Please contact your Cytek Technical Application Scientist and they'll be able to have a discussion with you and answer all of your questions.
@JimboMahoney64 жыл бұрын
This is a great little tutorial, thanks!
@UChicagoFlow4 жыл бұрын
Thanks, that's good to hear! Let me know if you have questions!