Ex Vivo Cytokine Analysis of Leukocytes

Cells were harvested from the ear and stained with fixable live/dead cell stain (ThermoFisher) (42 (link)). For ex vivo intracellular staining of cytokines, brefeldin A (1 μg/ml) was added to all media and wash buffers to block cytokine secretion (43 (link)). Leukocytes were stained with Abs (Biolegend) to CD45 (30-F11), CD4 (RM4-5) and Thy1.1 (H1S51), fixed with cytofix/cytoperm (BD Biosciences) and stained with anti-IFNγ Ab (XMG1.2). Analyzed by FACS using the BD LSR II.

Free full text: Click here

Fernandes N.R., Reilly N.S., Schrock D.C., Hocking D.C., Oakes P.W, & Fowell D.J. (2020). CD4+ T Cell Interstitial Migration Controlled by Fibronectin in the Inflamed Skin. Frontiers in Immunology, 11, 1501.

Publication 2020

fixable live/dead cell stain cytofix/cytoperm BD LSR II

Block Brefeldin a Buffers Cell Cytokine Ifnγ Intracellular Leukocytes Secretion

Corresponding Organization : University of Rochester Medical Center

Other organizations : University of Rochester

Top 5 similar protocols

Variable analysis

independent variables

Harvesting cells from the ear

dependent variables

Cytokine production (IFNγ)

control variables

Fixable live/dead cell stain (ThermoFisher)
Brefeldin A (1 μg/ml) added to all media and wash buffers to block cytokine secretion
Staining with Abs (Biolegend) to CD45, CD4 and Thy1.1
Fixing with cytofix/cytoperm (BD Biosciences)
Staining with anti-IFNγ Ab (XMG1.2)
Analysis by FACS using the BD LSR II

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!