Published: Vol 5, Iss 11, Jun 5, 2015 DOI: 10.21769/BioProtoc.1496 Views: 18322
Reviewed by: Ivan ZanoniAnonymous reviewer(s)
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
Related protocols
Protocol to Isolate Germinal Centers by Laser Microdissection
Farbod Bahreini [...] Kathrin Kalies
Jun 5, 2022 1865 Views
Immunohistochemistry of Immune Cells and Cells Bound to in vivo Administered Antibodies in Liver, Lung, Pancreas, and Colon of B6/lpr Mice
Kieran Adam and Adam Mor
Jul 20, 2022 2712 Views
Monitoring Group 2 Innate Lymphoid Cell Biology in Models of Lung Inflammation
Jana H. Badrani [...] Taylor A. Doherty
Jul 20, 2023 1874 Views
Abstract
Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (brand name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies.
We use a CyTOFTM mass cytometer to acquire the ICP-MS (inductively coupled plasma mass spectrometry) data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system.
Materials and Reagents
Equipment
Procedure
Representative data
Notes
Recipes
Acknowledgments
This work was supported by grants S10RR027582, 5U19AI057229, and 5U19AI090019 from the U.S. National Institutes of Health.
References
Article Information
Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Fernandez, R. and Maecker, H. T. (2015). Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry. Bio-protocol 5(11): e1496. DOI: 10.21769/BioProtoc.1496.
Category
Immunology > Immune cell staining > Immunodetection
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link