August 31, 2012  |  Cytobank  |  By  |  0 Comments

Dose Response Analysis

Dose response analysis on data acquired with a single cell level of resolution can provide important insights into the behavior of specific cell subtypes that cannot be gleaned from bulk population studies. See the image below to compare dose response and EC50 values observed in bulk whole blood versus CD8+ T cells. Krutzik et al showed this in earlier work using signaling-based flow cytometry [1]. A recent article by Bodenmiller et al further demonstrates the power of dose response analysis in highly multiparametric datasets generated using mass cytometry (see Cytobank Report here) [2].

Dose Response Analysis of bulk PBMC compared to CD8+ T cells reveals the power of single cell resolution and mining data specific to subpopulations


A key challenge in these works is generating and navigating complex dose response data linked to the underlying raw fluorescence data.

When you run a dose response flow cytometry experiment, you can use Cytobank’s Dose Response Analysis functionality (available on Enterprise Cytobank and Fluidigm Cytobank) to generate a dose response curve and determine the EC50, as well as specify a number of statistics associated with the data. This built-in functionality will help you translate your raw flow data into dose response curves, and can even be useful in plotting titration data without transitioning to an external analysis program. Being able to do standard flow cytometry analyses and dose response analysis within the same framework means that dose response data will remain linked to the raw data. You’ll be able to update and generate new plots by selecting additional parameters to display (e.g. additional populations, additional conditions). Any changes you make to analysis of the raw data (e.g. gating strategy) will automatically be reflected in the updated dose response plots.

The setup process isn’t too different from setting up Illustrations in general on Cytobank. You’ll begin by annotating your data: gate your Populations, make sure your Channels are labeled, and tag your FCS files with any other relevant descriptors such as Conditions, Timepoints, and Individuals. In this case, you’ll want to enable the Dosages figure dimension and tag your files with the corresponding dose.

Next, set up your illustration as if you were generating a histogram overlay. In the example below, we’ve grouped samples by Channels in the columns, Dosages overlaid, then further grouped by Populations in the outer table columns, and Individuals in the outer table rows.

Finally, click “Dose Response View” on the left side of the Working Illustration page, fill in your dose values in the Doses Dimension box on the left side of the page, update the illustration, and voila: your dose response plots and accompanying statistics will appear. You can configure the statistics under the Plot Controls section on the left side of the page.

Dose Response View

Because the dose response plots are directly linked to the underlying data, you can mouse over a dose plot and see the underlying data displayed as a histogram overlay:

Put your mouse over Dose Response Analysis plots to reveal raw data presented as histogram overlays

We have a number of Knowledge Base articles available to help you get started using Dose Response Analysis on Cytobank.

– Angela

References:

1. High-content single-cell drug screening with phosphospecific flow cytometry. Krutzik PO, Crane JM, Clutter MT, and Nolan GP. Nat Chem Biol. (2008) 4(2):132-42. [PubMed]

2. Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Bodenmiller B et al. Nat Biotechnol. (2012) Epub ahead of print. [PubMed]