The isolation of peripheral blood mononuclear cells (PBMCs) and flow-based transmigration assays were performed in a 3D BioFlux flow chamber device (Fluxion Bioscience, San Diego, CA) as previously described.30 (link) The migrated cells were enumerated by a hemocytometer and then normalized to migrated cell numbers determined by flow cytometry. After collection, cells were fixed for 10 minutes in 1% paraformaldehyde at room temperature and washed in phosphate buffered saline + 0.1 mM ethylenediaminetetraacetic acid, followed by blocking in mouse IgG. Cells were labeled with anti-human CD45 eFluor450, CD8a APC-eFluor780 (eBioscience, San Diego, CA), CD3Alexa Fluor 647 (BioLegend, San Diego, CA), CD19 BV711, and CD4 PE-CF594 (BD Biosciences). Data were acquired using BD FACSCanto II (BD Biosciences) and analyzed by FlowJo software (v.10.4.1; Treestar, Ashland, OR).
Protocol detail
Isolation and Flow Cytometry of PBMCs
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Assays
Cells
Device
Ethylenediaminetetraacetic acid
Flow cytometry
Human
Isolation
Mouse
Paraformaldehyde
Peripheral blood mononuclear cells
Phosphate
Saline
Corresponding Organization :
Other organizations : Chugai Pharma (United States), Third Dimension Technologies (United States), Yamaguchi University
Top 5 similar protocols
Variable analysis
independent variables
- Isolation of peripheral blood mononuclear cells (PBMCs)
- Flow-based transmigration assays
- Migrated cell numbers determined by flow cytometry
- Migrated cells enumerated by a hemocytometer
- 3D BioFlux flow chamber device (Fluxion Bioscience, San Diego, CA)
- Fixation of cells in 1% paraformaldehyde for 10 minutes at room temperature
- Washing in phosphate buffered saline + 0.1 mM ethylenediaminetetraacetic acid
- Blocking in mouse IgG
- Labeling with anti-human CD45 eFluor450, CD8a APC-eFluor780, CD3 Alexa Fluor 647, CD19 BV711, and CD4 PE-CF594
- Data acquisition using BD FACSCanto II and analysis by FlowJo software (v.10.4.1)
- No positive or negative controls were explicitly mentioned in the provided information.
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.
Sign up for free or login to display all annotations
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!