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Optimizing Instrument Settings for Flow Cytometry


You took great care in the design of your multicolor staining panel, taking into consideration expression levels of your antigens of interest, potential compensation issues…..all those issues you have been trained to consider! Never-the-less, when you actually ran your sample, some of the compensation values were much higher than you expected and the data looked a bit messy – what’s up with that? Using fluorescent beads to calibrate your instrument and set ‘optimal’ PMT voltages does not always translate to ‘optimal’ instrument settings for the particular staining panel you are using. In our example below, the staining panel includes a PerCP-Cy5.5 conjugate (B220) and a PE-Cy7 conjugate (CD3). As can be seen in the figure below, there is some overlap in the emission spectra for these fluorochromes that will have to be dealt with through proper compensation.

PE-Cy7 and PerCP-Cy5.5 Emission Spectra

Using the settings provided by the bead setup for our instrument, the voltage applied to the PerCP-Cy5.5 detector is 626 and for the PE-Cy7 detector, 669 volts. With this setup, our B220 PerCP-Cy5.5 single stained control looks like this:

PerCP-Cy5.5 Detector and PerCP-Cy5.5 Spillover in PE-Cy7 Detector

PerCP-Cy5.5 DetectorPerCP-Cy5.5 Spillover into PE-Cy7 Detector









YIKES! You know just by looking at this that a significant amount of compensation is going to be required to remove the PerCP-Cy5.5 signal from the PE-Cy7 detector. In fact, that number is 80% and once that is applied, the 2-color plot of B220 PerCP-Cy5.5 vs. CD3 PE-Cy7 looks like this:

CD3 PE-Cy7, B220 PerCP-Cy5.5

Of course, both populations are resolved but there is a lot of spread of the CD3 negative population. By simply decreasing the voltage to the PE-Cy7 PMT to 520, the compensation required to remove the PerCP-Cy5.5 signal from the PE-Cy7 detector is reduced to 12% with excellent resolution of both populations as shown here:

Improved CD3, B220 Data

Believe it or not, these data were collected from the same sample of stained cells, the only difference was optimizing the PMT voltage setting for the PE-Cy7 PMT in order to optimize resolution for this particular staining panel!

Some general thoughts:

  • Before you collect and save any data, open a screen with a histogram panel for each fluorochrome in your panel. Run each of your single stained controls and evaluate the spillover from that fluorochrome into all of the other open detectors. A good rule of thumb is to be sure that fluorescence in the correct detector is at least half a log brighter than spillover fluorescence in any of the other detectors.
  • Using fluorescent beads to calibrate your instrument will provide an excellent starting point for instrument voltage settings but optimizing for your particular panel of reagents will provide excellent data!


Yes, it’s true that right fluorescence detector can give you single stained control. Currently, I am using Stratedigm’s flow cytometer, but before that, it was tough to get results from single stained cell analysis. I tried to change the instrument settings, but failed. Anyways, I had been using that instruments since many years, and I wanted an advanced customized flow cytometry instrument. I configured S1000Exi flow cytometer as per my requirement, and I am overwhelmed with the results.
Posted @ Wednesday, December 11, 2013 4:10 AM by
Thanks for the nice blog! 
Your plot looks much better with decreasing your PMT voltage. 
But, I am doubting, in terms of loosing information and discrimination efficiency, are you really a step forward by doing this? Are you really increasing your resolution? When you lower your PMT voltage in the channel with the overlapping signal, of course your percentage of spectal overlap will drop, but the percentage of the signal in each channel which comes from the ‘wrong fluorochrome’ remains the same, or not? Regarding the high spreading of the negative population; it seems logical to me that if you lower the PMT the spreading of the negative peak also decreases, but is the discrimination efficiency really improved?  
Kind regards, 
Ben Meijer 
Posted @ Wednesday, January 22, 2014 3:07 PM by Ben Meijer
Hi - 
Great question, and thanks for the feedback! 
In the top row of this image, with unoptimized instrument settings we have a compensation value for PE-Cyanine5.5 out of the PE-Cyanine7 channel of 80.7%; when we optimize the settings by lowering the voltages, as seen in the bottom row, the compensation of PE-Cyanine5.5 out of PE-Cyanine7 drops to 39.6%. Additionally, when the instrument settings have been optimized, you can see that although the absolute brightness of the CD14+CD45+ cells is less than when we use the unoptimized settings, we actually have greater separation between the CD14+ and CD14- cells. If we were to replace CD14 with a marker that had a broad range or dim expression levels, then the ability to accurately quantitate the dim events (in terms of both percentage and MFI) would be greatly improved by using the lower voltage settings. 
Let us know if you have any other questions or comments! Thanks again! 
- Castle, Scientist, R&D, eBioscience
Posted @ Thursday, February 06, 2014 10:19 AM by Diane Gaige
Hi, great blog. it was nice to know how changes in voltages can actually improve compensation.  
I have a query. I have Navios (just got two Navios previously I was using FACS Canto-II),NOw started using 9-10 color. So I am getting a bit difficulty in setting satisfactory compensation especially with APC, ALexa700 and 750. So my query is " can we modify voltages for each tube and save them in that protocol or experiment and then apply tube specific compensation?". 
What are your inputs? 
Posted @ Sunday, March 02, 2014 8:58 PM by Prashant Tembhare
Great question! 
Compensation can be set only after all voltage settings have been established. Every time you change the voltage settings you are changing the relationship between the detectors, and, as a result, you change the compensation values required. This means that for a given experiment, you cannot and should not adjust voltages for each tube to get the compensation you want. As noted above, you should set voltages so that the signal you observe in the actual detector for a given fluorochrome is at least 0.5 log brighter than the spillover observed in all other detectors. Also note that different vendors have different methods for conjugation that can result in different compensation values, and this is especially true for tandem fluorochromes. So, APC-Cyanine7 is not the same as APC-Alexa Fluor® 750 nor APC-eFluor® 780, and each of these dyes will require specific and unique compensation values, despite being detected in the same channel on the cytometer. If each tube using the exact same fluorochrome-conjugated reagent is requiring different compensation values, be sure that you are protecting all of your samples from light and that they have all been treated the same after staining with antibodies (e.g., fixation). Exposure to light or different treatments can also result in different compensation values. 
Let us know if you have any other questions or comments! Thanks again!  
- Castle, Scientist, R&D, eBioscience
Posted @ Thursday, March 06, 2014 12:31 PM by Diane Gaige
Hi, is it also possible to increase the voltage of the channel that spills in the other one (in this case PercpCy5.5) to reduce the compensation values? and if yes, why? how do you explain that? Thank you for your reply. best
Posted @ Thursday, March 27, 2014 10:14 AM by virginia
Thanks for the great question! 
You should set voltages so that the signal you observe in the actual detector for a given fluorochrome is at least 0.5 log brighter than the spillover observed in all other detectors. This can be accomplished by decreasing the voltage setting for PE-Cyanine7 as already stated above or by increasing the voltage setting for PerCP-Cyanine5.5. However, if the signal for PerCP-Cyanine5.5 is already high (in the last decade), you may not have a lot of room to increase the voltage setting. The most important thing to have is at least 0.5 log greater signal intensity in the actual detector channel as compared to the uncompensated signal in all the other channels. 
Let us know if you have any other questions or comments! Thanks again!  
- Castle, Scientist, R&D, eBioscience  
- Emily, Technical Support, eBioscience
Posted @ Thursday, April 03, 2014 2:44 PM by Diane Gaige
Changes to voltage settings on the instrument will certainly affect the spread or “boundary” of the positive and negative cell populations. In the dataset above, we are comparing the differences in the spread of the PerCP-Cy5.5 signal into the PE-Cy7 channel. By simply lowering the voltage in the PE-Cy7 channel (669 top dot plot to 520 bottom dot plot), we see that the spread is decreased, creating a tighter population or less spread.
Posted @ Tuesday, January 27, 2015 5:46 PM by Garima Mehta
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