April 9, 2011  |  Cytobank, Education, Flow Cytometry

Teaching Phospho-Flow… in France

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.

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February 25, 2011  |  Cytobank, Education, Flow Cytometry

Immunophenotyping Tumor-Infiltrating T cells

Dataset #4659: Testing Set – T Cell Immunophenotype (trimmed)

Quantifying the percentage of cells expressing a protein of interest is a frequent goal in both basic research and clinical studies. Paired with per-cell comparisons of the level of protein expression, this approach provides a powerful way to track and immunophenotype populations of cells present in a particular sample.

One widely recognized application of flow cytometric immunophenotyping is determining the percentage of CD4+ cells in a gated lymphocyte population in order to determine prognosis for an HIV patient. Other applications include measuring a series of markers in order to distinguish between different forms of leukemia.

In Cytobank, you can use the “percent in gate” statistic to measure and display the percentage of cells in a selected gate as compared to each active population in your figure. To illustrate with a simple example, let’s examine a sample dataset looking at the percentage of CD25+ cells in a CD3+ T cell population.

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December 21, 2010  |  Cytobank, Flow Cytometry

Working with Compensation in Cytobank

Let’s just say it: compensation is a pain. Unfortunately, it’s a necessary pain if we want to accurately interpret our flow data and draw meaningful biological conclusions.

As a brief reminder of why we need to care about compensation, let’s examine the following emission spectra. We used the BD Fluorescence Spectrum Viewer; the Fluorescence SpectraViewer by Invitrogen/Life Technologies is also useful.

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December 14, 2010  |  Cytobank, Education, Flow Cytometry

Biochemistry at the Single Cell Level

Dataset #811: 090902-PBMC stim with IL6, IL10, LPS (Public Version)

Western blots are great. That is, unless you’re studying signal transduction in a mixed population of primary cells. The amount of material necessary for many biochemical techniques is usually not practical when working with patient samples or rare cell populations.

Flow cytometry to the rescue! Phospho-flow tracks biochemistry at the single cell level. Simultaneous assessment of cell surface markers and signaling events for individual cells enable biologists to profile disease samples or monitor the effects of drugs in clinical samples. More »

November 17, 2010  |  Cytobank, Flow Cytometry

Take the U937 Challenge!

Dataset #61: Welcome to Cytobank – U937 dataset

So you want to do phospho-flow? Take the U937 challenge!

What is phospho-flow? It’s a specialized form of flow cytometry that enables biologists to probe signaling activity inside the cell. Flow cytometry has been widely used to analyze cell populations by probing the proteins that are outside on the cell surface. Phospho-flow cytometry includes a few extra steps – stimulation, fixation, and permeabilization – that enable researchers to also probe the transient phosphorylation state of intracellular signaling proteins. More »