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Intensity of Normalization Beads

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Please be as geeky as possible. Reference, reference, reference.
Also, please note that this is a mixed bag of math-gurus and mathematically challenged, so choose your words wisely :-)
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billog

Participant

Posts: 15

Joined: Wed Sep 17, 2014 10:53 pm

Post Tue Mar 07, 2017 5:41 pm

Re: Intensity of Normalization Beads

Hello,

I just wanted to 2nd Adeeb's comments as we also found that the Fluidigm fix/perm buffer when used for the final fixation did not produce stably fixed cells and signal loss from robust markers such as CD3 and CD19 was subtle yet apparent even after 30mins of a sample sitting in 100% H20 and very obvious after 6hrs of 100% H20 exposure. We took one sample ran it immediately after removing it from PBS/Fluidigm fixative/IR and it looked great but that same sample after sitting in H20 for six hours would have obvious signal loss and even increased background in some cases. We compared this head to head with using EMS 16% PFA from freshly opened ampules and this was far superior, I would say EMS PFA approach is "good enough" but more testing would need to be done to ensure absolute stability of a number of markers. Bottom line for me was do not use the Fluidigm fix perm buffer at least in conjunction with the Ebio Human Foxp3 kit that we were using prior to the final fix with the Fluidigm fix/perm buffer and as long as your EQ beads are fresh and give you consistent data (meaning matlab normalization aligns your signal intensity from your beads at different time points in your run) then don't immediately suspect you have a hardware issue. The 1st suspect should be the quality of your fixative and fixation. Trust the beads. I do believe however that the problem with the Fluidigm fix/perm buffer is more generalizable than simply being an issue when used in conjunction with the Ebio Human Foxp3 approach.

I sent this data to Fluidigm quite awhile back, some action was taken on their part to reproduce these results but I'm not sure were it went from there... Sorry I should have raised this issue on this forum earlier.

- Bill
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FlowjoVA

Participant

Posts: 12

Joined: Tue May 24, 2016 4:49 pm

Post Tue Mar 07, 2017 11:51 pm

Re: Intensity of Normalization Beads

Thanks Adeeb! I suspect you are right about this.We plan to test the fixative issues you refer to, thanks for the advice. If this turns out to be the case I would think it prudent that Fluidigm include this information in their protocols and package inserts. We are also evaluating the effect this has on data clustering algorithms. Stay tuned!
Thanks everyone who provided very valuable input.
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SteinErik

Participant

Posts: 3

Joined: Fri Sep 30, 2016 6:18 am

Post Mon Jul 10, 2017 10:47 am

Re: Intensity of Normalization Beads

Hello,

Thank you all for the very interesting discussion. I also see similar loss of antibody signal relative to the normalization beads. In a recent experiment, I had all my cells barcoded into one antibody stained pool. The pool was split into 6 aliquots. Each aliquot was washed (2x CSM and 1x MP H2O) and pelleted just before start of each acquisition. Sample acquisition ranged from 15 min to less than 30 min.

The bead normalization does not correct sufficiently for temporal loss of antibody signal in each aliquot. As all my cells are barcoded into one pool, is it feasible to actually use a strongly expressed cell surface marker for normalization? (CD4/CD66b/CD123/CD3). I don't care about any global standard to compare/normalize my data to, i just need it to be stable over time in my barcode pool.

-Stein-Erik
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