Hi Matthew, Flow always gets a bit trickier when you get away from the small round cell paradigm! Vero cells will be larger and more epithelial than lymphocytes so the voltages used for FSC and SSC will likely be lower than if you are using lymphs and much lower than those set by any QC beads. If you are using a FSC v SSC dotplot be careful to see if there are any events piled up on the right or upper axes. You can bring these on scale by lowering the appropriate voltage. It is liley that there will also be much more of a spread of the cell 'cloud' than with beads or lymphs. If you are using a BD machine, you may be able to alter the ASF (Area Scaling Factor) - plot FSC-A v FSC-H - the cell population should have about the same value on each axis - if they do not you can alter the ASF to align them. Hope that helps!

@@DerekDaviesCytometry thanks so much! Super helpful!

Hi Hank! Yes, you can see all of our classes on our page. kzfaq.info Is this what you were looking for? Happy Flowing! - Kathy

Hi Akriti. There's a lot to unpack in your question. 1) In conventional flow cytometry there's a one-to-one relationship between fluor and channel, i.e., you pick the best channel that will detect a given fluor. Therefore, you don't have to worry about the voltages/gains in other channels. As we mentioned in a few of our sessions, first you optimize the voltages and gains of each channel in the cytometer. Then you titrate all your ab-fluor conjugates, and then you acquire every control and sample. The only reason to adjust voltages AFTER optimizing the voltages of each channel is when your samples or controls show signals of scale. Apart from that, you should leave ALL voltages/gains untouched regardless of spillover. Yes, even if the signal of a fluor is higher in a secondary (or spillover) channel than in the primary channel (the one you chose to detect that fluor). 2) Flow cytometry measurements are relative and displayed in arbitrary units. Since we can change voltages or gains, a positive signal can be anywhere in the scale. So signals above 10^3 can be either positive or negative, just like signals below 10^3 can be positive or negative. You must run a negative control to define what is positive and negative, and those signals can be anywhere in the scale. 3) I'm not sure I understood your final question, but if the antibodies were not well titrated, or your instrument was not well optimized (we call it "instrument characterization", which means optimizing your voltages/gains for each channel) you may not see a good separation between positive and negative populations. So the problem you seem to be facing (again, apologies if I'm not understanding your question fully) could be related to the instrument not being well optimized in the first place, and/or insufficient titration. Did this answer your question? Please feel free to elaborate and we'll be happy to answer.

@@RuiGardner Thank you for the quick reply! Yes, you have partly answered my query. But I still did not understand most of it (sorry!) I shall delineate as to what I do - 1. I prepare single stained controls, FMOs and unstained control using splenocytes while the samples that are stained fully would be meninges 2. At the cytometer (we use FACS CantoII here), this was what I was taught. i) First, to adjust/optimize my voltages, I run the unstained at low and check if the peaks of all the other colours are below 10^3 (without recording). ii)Then, I run every single control individually (without recording ofc) to set the voltage in order to ensure that the single stain control in question is beyond 10^3 (or brighter for positively stained) and for rest of the colours at the same time, their peaks need to be less than 10^3 log scale. I consume alot of time here because certain colours like PCP have spill overs into APC, PE Cy7 and even AF700 channel on Canto II. Therefore, I ended up spending a lot of time in reducing APC's, PECy7's and AF700's voltages only to ensure that when I add PCP single stained tube at SIP, I must get (idealy, I think) a positive peak beyond 10^3 with the adjust voltages! what I also came across was that therenwere small peaks in the other 3 channels were beyond 10^3 (just like in PCP's channel) while running PCP single stained tube (and I ensured that the single stained wells weren't conatminated by other colours!) Am I doing it right? Also, I could not follow up on the difference between adjust and optimizing the voltage that you have stated above in the answer. Please pardon me if I am asking a silly question!

@@akritino7437 Agreed with all that Rui said! Also, not a silly question by you at all! We often see that researchers are trained in this way based on older ways of thinking for instrument setup. The way that you are explaining is only going to cause you confusion and headaches. One of the best approaches for setting voltages is to run your single stain controls at increasing voltages. Since you are working with the splenocytes, I imagine that you have a positive and a negative population for most all of your single color controls. You record files at increasing voltages until you see that the signal is off scale (Just beyond 10^5). You can then calculate the Stain Index for all samples to determine the voltage that gives you the optimal separation of positive and negative. We go over this in depth here: kzfaq.info/get/bejne/jM6ljaeBu56ZeIk.html Now as far as decreasing other detector voltages in order to minimize spillover, this is not necessary! Remember, you want to optimize to get the best separation. If you start decreasing voltages, you will reduce the separation between positive and negative and risk the potential of not seeing dim or lowly expressing markers. Some people are worried about compensation values over 100%. That is misguided. If you look at this Flow Post-It by Barbara Oliveira you can see that adjusting voltages to lower compensation does NOT mean you get a better separation of signal. wi.mit.edu/sites/default/files/2021-12/20211004%20SOV%20over%20100_0.pdf I suggest you watch the voltage optimization OpenFlow and follow the guidance there to set your voltages. Though I will add that if your experimental sample is a different cell type than the sample you are working with for your single color controls, there may be a large difference in autofluorescence. If your experimental sample has higher autofluorescence, this can impact the optimal voltages, and you may need to reassess with the cell type of interest, taking into account the impact that difference in autofluorescence has on the separation. Best of luck, and please let us know if this all makes sense! Kathy

@@RuiGardner Okay, so I have seen your videos just to get my doubts further clarified. So, in a nutshell, just to be sure that I have gotten the concept right are the following - 1)For instance, if a voltage set is appropriate for particular colour, say PE, we must choose that voltage inspite of the fact that other colours could interfere with their signals popping up beyond 10^3 at that set volatge. Am I right? 2) So, the negative of any colour is based on the unstained population. So depending upon where our unstained population has been set, one doesn't have to bother about the decades on the log scale as long as they (unstained cells' curve) remain on the left hand side of the graph. Am I right again? 3. I used to previously assume that voltages could correct the compensation values. i.e. If I choose a voltage for a particular colour, A, at say 450, that voltage must be adjusted in such a way that the interfering signals from other colours at that voltage must not exist beyond certain voltage (10^3 since below this log decade, we had set the voltage of unstained). This adjustment would consequently reduce the compensation. That was what I had previously thought of. Therefore, it could be right to say that adjusting voltage does not necessarily reduce compensation. Rather, it simply changes the values of compensation. Am I correct over here? Last question. I agree that the autofluorescence of single stained control cells, unstained cells and our sample cells need to be the same. However, if one is using splenocytes to optimize voltages and compensate only and other cell types as samples, one could slightly change the scatter voltages of the sample while running. Is this correct? Or is there is any limitation to this? A point that I understood (and what my mind extrapolated from what Dereck stated in the video), was that if an unstained control is replete with Mac/DCs like in one's sample (even if the two belong to different organs), that is still fine as long as te autofluorescence remains more or less the same. Would this be the limitation? Am I making sense? That 's all I have got so far ! Please correct me if I have erred anywhere here! I just want to get my concepts right and be confident!

@@akritino7437 Hi Akriti, Many thanks for your great questions and hopefully our answers and other videos have helped you. But in answer to your questions: 1. It is very important - in fact perhaps the most important part of your experiment - to set the correct voltage for any particular channel. We then deal with overlap by using compensation but the value of compensation shouldn't be a primary concern. If you find that one colour is brighter in another channel than that channels fluorochrome then it may mean that this is not a good combination of fluorochromes. 2. As long as the positive and negative are as far apart as possible and the positive population is not above 10^5 then it doesn't really matter where the negative population is. We should get away from the idea that our negative population should be right in the bottom left corner of the plot. 3. Changing voltages will change the compensation values But I would caution against doing this because, particularly if you are reducing the voltage, you will be lowering sensitivity and potentially missing weakly positive cells. No matter what the value for compensation though the spread of data ,which is the thing that will reduce resolution in the spillover channels ,will remain the same. 4. It is perfectly acceptable to change the scatter voltages during running these do not affect compensation. The concept of ensuring you compensate to an unstained autofluorescent population becomes important in heterogeneous cell populations. If you had a mixture of say monocytes and lymphocytes and you compensate your monocyte marker to the unstained monocytes level of autofluorescence and lymphocyte marker to the unstained lymphocyte level of autofluorescence then the compensation will be equally applicable to each cell type as long as you haven't changed any voltages! Let us know if you still have any outstanding questions.