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
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.
How to Justify an iPad in Your Grant Cytometry in the Cloud
Fill in your details below or click an icon to log in:
You are commenting using your WordPress.com account. ( Log Out / Change )
You are commenting using your Twitter account. ( Log Out / Change )
You are commenting using your Facebook account. ( Log Out / Change )
You are commenting using your Google+ account. ( Log Out / Change )
Connecting to %s
Notify me of follow-up comments via email.
Trackback this post | Subscribe to the comments via RSS Feed
Enter your email address to subscribe to the Cytobank blog and receive notifications of new posts by email.
Join 13 other followers
RSS - Posts
Blog at WordPress.com. Customized Blix Theme.