Cytobank – As the cell flows

Teaching Phospho-Flow… in France

April 9, 2011 at 11:42 am Stephanie Huang Leave a comment

Dataset #5002: Timecourse LACI 2011

This January, Jonathan and Chris from Cytobank traveled to Marseilles, France to help lead a course as part of the Luminy Advanced Course in Immunology (LACI). LACI is organized as a satellite meeting to the Immunology and Metabolism meeting and organized by the Centre d’Immunlogie de Marseille-Luminy (CIML) and the European Molecular Biology Organization (EMBO).

The ‘Cell Signaling’ course at LACI was taught by local instructors Nathalie Auphan-Anezin and Pierre Grenot, both of CIML, and Jonathan and Chris. The course led course participants through staining, collection, upload, and analysis of a phospho-flow experiment. We’ve briefly described the experiment here, made a version of the dataset public along with the original course protocol, and prepared a tutorial (part 1 and part 2) to lead you through Cytobank analysis of the course data.

The participants started with stimulated primary mouse lymph node cells which were comprised primarily of B and T cells. Cells were stimulated with either IL-7 or IFNα in a reverse timecourse ranging from 0 to 45 min. Following stimulation, cells were fixed (to stop signaling and preserve the phosphorylation status of signaling proteins) and permeabilized (to enable staining antibodies to bind to intracellular targets). The cells were stained with antibodies against CD44, CD4, TCRβ, B220, p-STAT5, and p-STAT1. B cells were identified by positive B220 staining, and T cells were identified with positive CD4 staining. Phosphorylation of STAT5 and STAT1 was measured as a read-out for IL-7 and IFNα signaling, respectively.

Stimulation by IL-7 or IFNα activates specific signaling pathways in different cell subsets. For example, only the T cells are expected to signal following IL-7 stimulation in this experiment. The B cells provide an internal control.

Here’s an example of data from the course, collected by Group 3. The histogram overlay shows p-STAT5-Alexa488 signal in IL-7 treated T cells and B cells.

Download the full experimental protocol, which includes a list of the reagents used.
Interact with the public dataset

Watch a video tutorial of analysis of data from this experiment

• Part 1: Cloning, Channel Annotation, Gating
• Part 2: Timepoint and Condition Annotations, Rearranging Dimensions, Saving Illustration

Questions or comments? Email us or comment below.

- Jonathan and Stephanie

Entry filed under: Cytobank, Flow Cytometry. Tags: CIML, data analysis, flow cytometry, histogram overlay, illustration, LACI, Marseilles, phospho-flow, phosphorylation, video tutorials.