Does anybody know how I can correctly adjust the compensation matrix for multicolor flow. I used the BD Comp beads to set up the matrix in FlowJo, yet it doesn't fully compensate my samples.
I didnt realize it at first (my first time doing this) and processed my samples.
I have the FMOs, but not the single color controls for my samples (I wont be able to do them since I run out of the stock antibodies, I might be able to if I really really get lucky)
If anybody could recommend how I can set it up, and adjust the matrix in FlowJo given what I have, that would be greatly appreciated.
Hi Aleks,
single color controls of your sample are much better to compensate your experiment compared to beads. And they are imho more important than FMOs.
I would say that compensate a data set later in FlowJo without proper single color controls could be tricky - as you've already experienced. For multi-color-FACS FlowJo offers a nice automatic compensation option (what I would prefer over manual compensation), did tried that with your comp beads?.
Best
Thomas
Hi Aleks Thomas is right about his suggestion. However, we are unsure what are you measuring in which population. If it is lymphocytes then use the program if it is in ganulocytes or monocytes then you do have to compensate using a proper control and block unspeficif binding using 1 % AB human serum for 10 min at room temp wash and add the antibody coctail. I hope it is useful
Hi Thomas,
It looks like I have enough antibody stock to run one or two sets of single stain controls. (I dont think i need to run the All color samples...)
I am looking at the leukocytes population in the mouse brain after the inflammation.
I am trying to identify the subsets of this population, as well as determine and compare the TNFa levels between them
The all color staining seems to be working good. The problem I have is the single color samples.
These cells seem to have high autofluorescence and i cant seem to get a clear separation between the positive and negative populations. I tried to use FlowJo compensation wizard to set up the matrix, but it doesnt recognize the positive population??!! Thats why I turned to the Comp Beads..
As I meantioned earlier, the compensation matrix based on Comp Beads doesnt seem to compensate my samples well .
I am wondering what choices do i have left.
Since i initially thought I had my comp matrix set correctly, I processed my real samples. These samples are extremely valuable and I hope there is a way for me to set up the compensation matrix to analyze them
If I get single color controls from the cells I am working with, I am wondering if I can do manual compensation.
Although, I do not know all the caveats of setting up the manual compensation, is it something realistic to do. (i know its a lot of work, but given the circumstances, I need to read the data from my samples)
If you have any suggestions, please let me know
Thanks
Hi Juan,
I am looking at the leukocytes population in the mouse brain after the inflammation.
I am trying to identify the subsets of this population and determine and compare the TNFa levels between them.
The cells seem to have high autofluorescence and the single stain controls of these cells dont give me a clear distinction between the negative and the positive cells for the Flowjo to compute the matrix
Yes, I use Fc block for non specific binding
Please read what I wrote above to Thomas about my issue, if you have any suggestions, please let me know
Thanks
Hey Aleks,
I would be more than glad to help you with setting up your compensation matrix or looking at your data with you on FlowJo to setup a matrix using our algorithm. Contact us at [email protected] or here for more help, include your FCS files.
Also, you might take a look at our documentation on how to setup a comp matrix or drmr.com for more info on compensation matrices.
Thank you,
Miguel
FlowJo Tech Support
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