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Lower convolution threshold - how low or high it can be

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prybakowska

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Posts: 4

Joined: Sun Nov 06, 2016 9:56 pm

Post Mon Dec 05, 2016 9:38 pm

Lower convolution threshold - how low or high it can be

Hello Cytofers,
I wanted to start subject about lower convolution threshold. I am working on Cytof2 upgraded to Helios. I know that a default setting is 400, however it will depends also on the amount of channels that we will open in the particular experiment, right?
My question is how you are dealing with this?
I have one experiment when I am measuring cytokines response, so my panel for now is about 20 markers, however I would like to apply something like MMM (mass minus multiple) control in order to see how to put the gates, so in the panel of MMM I would have 9 AB less. I assume that I would need to change the lower convolution threshold, as I will have less channels open?
And is playing with the threshold really helps with getting rid of the debris and instead acquiring more data?
I have also heard that sometimes people are using threshold around 1000 which compare to the one suggested by fluidigm sounds high…. I have acquire few samples with around 20 markers (including cytokines) at 1000 and now I am wondering if that was OK and if by using IMD files I can still re-analyze my data at let’s say threshold 400. And if I do so, how exactly I can verify if one or the other threshold works better ?

Best,
Paula
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mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Dec 06, 2016 4:46 pm

Re: Lower convolution threshold - how low or high it can be

Hi Paula,

To be honest, I don't mess with the Lower Convolution Threshold default setting.

The only times I've messed with it have been during some control experiments with Capture beads (Bangs, BD kappa capture, etc), where I'm only using 1-2 antibodies at a time, and have varying signal intensities (thereby causing strong variations in Event Length, since EL is driven primarily by total metal content). During regular cell-based experiments, I just use the default.


It would be nice if Fluidigm would explain in greater detail just what some of these settings are, especially since, looking at v1/v2/Helios manuals, the default appears to change from model to model of the instrument. I commented on a separate thread yesterday (viewtopic.php?f=4&t=582) about finally getting an explanation from Fluidigm about what Noise Reduction actually does.


Mike

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