What organ is that ? Looks weird.
Fluorophore combination definitely not the best. Try to put your CD11b on a violet laser (Pblue orange or BVs).
Have you done any kind of dead cell exclusion? The compensation looks off as well, plus there might be a lot of dead matter.
Hi All,
Thanks for your message.
The sample is mice colon tissue, when I exclude the dead cells by gating on 7-AAD, the probelm is still exist. I try to analyze with different compensation, but when gating PE, PE/CYy7 and PE/Dazzle together, it always have the same problem with one color spills over into another color. How to solve this problem by compensation ?? Thanks!
You could post the single staining controls to see if compensation and signal-to-noise ratios are adequate. It could be many things (Autofluorescence, poor signal/high background, inadequate compensation).
Maybe your sets on filter to read these colors are not optimal...
Can you tell us your configuration :
LSR ?
blue or yellow green laser ?
filter and dichroic ?
The CD11b PE-Cy7 single stain looks ok (though you might want to bring the voltage down a bit to avoid losing your positives), but the CD11c PE-dazzle doesn't appear have a clear positive population to compensate with (though hard to tell from the dot plots, a histogram would be helpful). If you really must use these flurochromes (I assume you have other markers in other channels?), then it might be worth using beads to compensate with. Fc block and dead cell exclusion, along with better compensation control should clear up the plot quite a bit!
Hi Xin,
I am sorry I don't think I explained myself correctly. In all your samples (unstained and single stains), please show the PE-dazzle vs PE-Cy7 plots. Not the FSC vs SSC or the SSC vs the FLChannels.
Those are the ones needed to evaluate autofluorescence / background noise and compensation.
The fluorophore combination shouldn't be a problem (at least not on any modern machine) but If that's your comp control for PE/Dazzle you seem to be missing a positive population to comp with as noted by Amanda above . . . . If I have any 'questionable' stains (i.e. stains where I don't know if there are a significant number of positives in the sample OR where the staining intensity is in question) I ALWAYS include 'good' stains (e.g. CD4 when staining lymphocytes) conjugated to the appropriate fluorophores as well as the 'real' controls . . . it's not perfect if you have to use them for the comps but at least you have something 'close' to work with.
It appears that compensation on the PE-dazzle channel is insufficient. When you stain with PE-Cy7 alone most cells appear to have intermediate expression with a small population having a high expression and none of these intrude into the PE-Dazzle channel (thus compensation is ok). But when compensating the signal on the PE-Dazzle channel most cells above 10e4 are intruding into the PE-Cy7 channel. These PE-Dazzle Hi cells are the ones that appear to cause the trouble on your doubly stained sample.
Dear Xin Wang, I think that the problem is (at least partially) in the high degradation rate of the phycoerythrin-tandem dyes. It is known that an exposure to light, handling (centrifugation, fixation, high concentration of proteins, etc), storage conditions (temperature), multiple-step staining procedure, etc. lead to the tandem dye degradation. In your case, it will mean that if you bought CD11b (PE/cy7), you will always have same CD11b-PE (from many phycoerythrin-tandem dyes, PE-Cy7 showes the highest degradation rate). Usually, if you use simple (1 step) staining procedure, you should be able to compensate, however, if you use 2-3 steps staining procedure with cell fixation and permeabilization and intracellular staining, etc. it is not always possible to compansate (we tried a lot of different things, but the best option in our case was just to exclude or replace one of these dyes (PE or PE-Cy7) with another fluorochrome. Remember that during storage, long-term degradation rates are lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate can be decreased to 0.3%/month. Therefore, another option that you can think of, is to replace (buy a new one) your PE-Cy7 conjugated Ab. Success!
Hi Xin, yes, it could be that the CD11b-PE/Cy7 antibody that you have used are old or were stored not good (vor example at higher temperature or often exposed to light). In this case your PE/Cy7 tanden will degrade. As a result, in the vial you will have some of CD11b-PE/Cy7 Ab (let say 70%) and another 30% of Ab will lose "Cy7" and will become CD11b-PE. Very "simple" way to check your Abs it is to use the same staining that you used earlier (all steps), but in the same time do 3 stainings (one - CD11b-PE/cy7 + CD11c-PE/Dazzle594, another - only CD11b-PE/cy7, another - only CD11c-PE/Dazzle594). So you will able to compare these 3 stainings and will see if this CD11b-PE/Cy7 Ab give a lot of positivity in the PE-channel or CD11c-PE/Dazzle594 Ab give a lot of positivity in the PE/Cy7-channel. So you will able to see which Ab causes the gating problem. Then you can "fix" the problem by: buying a new vial of "bad" Ab (I guess that it will be the CD11b-PE/Cy7 Ab) or using the CD11b Ab with another fluorochrome (APC, Fitc, or something else that will be suitable and useful in case of you Abs combination and flowcytometry machine). If you need some advise, please feel free to ask (it will be easy to make a suggestion, you briefly describe your staining procedure and all fluorochromes that you use in your experiment). Success!
Hi Xin,
I would also change the biexponential display, in order to see also the cells on the "axis".
Hi,
some basic questions: what cells are you looking at? Spleen? Lung? ....
next: did you do FSCA-H gating to exclude debris?!
My opinion: you are looking at debris (autofluorescence peaking in diagonal) and therefore the compensation looks right; do you have the %comp matrix avaiable?
And if I look I think there are only CD11c cells which are CD11b positive and no single ones... (see attached picture), but as I said in the beginning.. there is some basic questions for me to say it more correct. If you like you can upload the 2-3 files, then the community could have a look at the raw data.. makes it more easy to give an answer :-)
I would also add a viability stain. It looks like there might be autofluorescent dead cells in your sample. There are several nice amine-binding ones to choose from
Hi Xin,
I don't use Flow Jo but but I suppose you can change the scale of the axis. The dot-plot presented in logicale allows you to modify the range of the axis value, for example in your dot-plot the value of fluorescence range from 0 - 10^5, when set in "logicale" you can re-adjust the scale the see the values from below 0, for example 10^-1, this will allow you to see the cells that have low fluorescence stuck below your axis, therefore you can present all the cells within the plot area.
Hi Xin,
The exported fcs-files must be version 3.0 or higher. Version 2.0 don't support Logicale/Biexponentioal display in FlowJo I believe. In some Cytometer Software , you might have do changes in the export settings. I mainly work with BD-instruments, and there it works by default, but fcs-files from other Software may be different.
Kerstin
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