I need some information whether it is possible to extend the logarithmic scale (x axis, MFI level) in flow cytometry histograms? In my case the maximal value is 104, which is to low for some samples. I can not manipulate voltages due to control and isotype control start to be invisible.
Thanks in advance for any suggestions.
Magda
Hello Magdalena,
I am not sure if your problem is in a data analysis program or with the analyzer itself. If the data is recorded in an analyzer then you cannot manipulate it afterwards, meaning the data doesn't change. It seems that the range of your analyzer is 10^4 which in most cases is perfectly fine. You are also saying that your control (negative population?) is invisible if you lower the voltage of that detector? You mean the population will go of the axis? I need more information from you to give a more specific answer. The log scale is a fixed range in the analyzer, usually with a analyzing program you can extend this fixed range, but it wouldn't look prettier. You can lower the antibody concentration?
Best regards,
Johan
Hello Johan,
thank you for response and suggestion. I try to use the lower concentration of antibodies.
Best regards,
Magda
There are two different considerations to be made on this topic:
1 - Employ the correct technique when acquiring your data
2 - Improve the visualization of your correctly acquired sample during analysis
For the 1st point, your should consider the following:
- select the antibody concentration used for staining with titration (there are many guides and protocols online). This allows to maximize the staining index, ie. the discrimination of positive vs negative cells.
- select the PMT voltage through a similar process to titration, not just by trying to set the "negative" population in the 10^2 - 10^3 range. The goal is to get the best separation of stained cells as in the case of antibody concentration. In any case, your positive cells/beads/etc should never be off scale or even outside the linear range of acquisition for your instrument. This translates to 10^4 and 10^5 for most cytometers, but you should run your own setup controls and find out what works best in your setting. If you have visualization problems during acquisition, be sure to select bi-exponential axis (ie. in BD Diva software) in the software and possibly adjusting the range (the latter may be necessary with specific instruments, ie. Beckman's Cytoflex).
For the 2nd point, if your data has been correctly acquired (ie. everything is within the dynamic scale of the instrument and linear range), you can manipulate the visualization in your software. This is done by changing the transformation applied to the data (ie. exponential, logicle, hyperlog, etc.. and the specific values used) to allow meaningful visualization of very low values (your negative populations, unstained or isotype controls) on the same plot as your stained cells.
Check FlowJo guide for biexponential transformation adjustment (https://docs.flowjo.com/flowjo/graphs-and-gating/gw-transform-overview/gw-transform-procedure/) or FCS Express (https://fcsexpressdownloads.s3.amazonaws.com/manual/manualV6_WIN_RUO/axis_options.htm). In FCS Express, beyond transformation selection and axis range, you can you the Magnify command to quickly change the visualization scale on the plots.
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