Identifying antigen-specific CD8+ T cells

Single-cell suspensions were first pretreated with anti-CD16/32 (2.4G2) antibodies to block Fc receptors and then stained with anti-CD11c (HL3), anti-MHC class II (M5/114.15.2), anti-CD317 (927), anti-Siglec-H (551), or anti-CD45.2 (104) antibodies. Live cells were gated based on 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) exclusion. Multiple cell populations were separated by flow cytometry (LSR Fortessa or Calibur; BD Biosciences). H-2D_b tetramers specific for the RSV M_187–195 peptide (NAITNAKII) were prepared with streptavidin-allophycocyanin (APC), using the protocol of the NIH Tetramer Core Facility (https://tetramer.yerkes.emory.edu/support/protocols) [17 (link)]. Single-cell suspensions from the lungs of immunized mice were pretreated with anti-CD16/32 (2.4G2) antibodies to block Fc receptors and then stained with anti-CD8α (53-6.7) and anti-CD3e (145-2C11) antibodies. Then, APC-labelled tetramer staining was performed. The number of RSV M_187–195 peptide-specific CD8⁺ T cells was analyzed by flow cytometry (Calibur; BD Bioscience). Final analysis and graphical output of the results were obtained using FlowJo software (Version 9, Tree Star, Inc., Ashland, OR, USA).

Free full text: Click here

Kim T.H., Oh D.S., Jung H.E., Chang J, & Lee H.K. (2019). Plasmacytoid Dendritic Cells Contribute to the Production of IFN-β via TLR7-MyD88-Dependent Pathway and CTL Priming during Respiratory Syncytial Virus Infection. Viruses, 11(8), 730.

Publication 2019

4’,6-diamidino-2-phenylindole (DAPI) LSR Fortessa Calibur

Allophycocyanin Antibodies Block Cd317 Cell Fc receptors Flow cytometry Lungs Mhc class ii Mice Peptide Peptide t Populations Siglec Streptavidin Tetramer Tree

Corresponding Organization : Korea Advanced Institute of Science and Technology

Other organizations : Changwon National University, Gyeongsang National University, Ewha Womans University

Top 5 similar protocols

Variable analysis

independent variables

Anti-CD16/32 (2.4G2) antibodies used to block Fc receptors
Anti-CD11c (HL3), anti-MHC class II (M5/114.15.2), anti-CD317 (927), anti-Siglec-H (551), or anti-CD45.2 (104) antibodies used for staining
H-2Db tetramers specific for the RSV M187–195 peptide (NAITNAKII) prepared with streptavidin-allophycocyanin (APC)

dependent variables

Live cell population gated based on 4',6-diamidino-2-phenylindole (DAPI) exclusion
Multiple cell populations separated by flow cytometry
Number of RSV M187–195 peptide-specific CD8+ T cells analyzed by flow cytometry

control variables

Single-cell suspensions from the lungs of immunized mice

positive controls

Anti-CD16/32 (2.4G2) antibodies used to block Fc receptors

negative controls

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!