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Gate threshold for cytokines

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Posts: 4

Joined: Sun Nov 06, 2016 9:56 pm

Post Sat Feb 11, 2017 5:37 pm

Gate threshold for cytokines

Hi Cytofers,
I am trying to verify the expression of the cytokines upon stimulation in the blood, I would like to perform standard gating to see by eye the expression level of the cytokines, I was wondering how to decide where the expression of the cytokine is starting to be true, in different words how many dual counts needs to be achieved to consider the cell positive and to setup the appropriate gate?
I was reading the paper:
Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry,
Michael D. Leipold, Evan W. Newell, and Holden T. Maecker. Methods Mol Biol. 2015; 1343: 81–95.
In there, it is said that everything below 10^1 is consider as a background, is 10^1 counts a good way to setup positive vs negative gate?
In the papers mentioned above there is an gating example for different cytokines but the thresholds for the gate differs a bit depending on the cytokines, can someone advice how to decide that one population is positive for IFNy at 10^2 vs for IL-2 at let’s say 60 dual counts? Or should I assume that my background is let’s say 50 dual counts for each and every cytokine?
I would be grateful if someone can explain how the gest were setup in this paper or in general how to setup them correctly.




Posts: 2162

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Feb 13, 2017 4:32 pm

Re: Gate threshold for cytokines

Hi Paulina,

The threshold depends on the background for each cytokine. Intracellular staining often has higher background than surface staining.

To a first approximation, you can set your positive gate above the signal intensity seen for your unstim sample, at least for a healthy control. Alternatively, you can set your positive gate above the signal intensity for a cell type that doesn't produce that cytokine (either at all, or under that particular stimulation condition).

Regarding background signal in general: yes, in general, the Dual = 10 signal intensity is usually a good place to start for dividing background from "true" signal. Others set the limit a bit lower: Takahashi et al set Dual = 5 as background, based on metal-minus-one experiments (http://dx.doi.org/10.1002/cyto.a.22977).

But as shown in the Leipold et al MMB paper, that Dual = 10-30 cut-off would be OK for GMCSF and IL-2, but too low for IFNg or IL-17.


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