Multiparameter Analysis of Immune Cells

Single-cell suspensions were prepared from the spleen, lymph nodes (LNs), or peripheral blood lymphocytes (PBLs), and surface or intracellularly stained as described (19 (link)). The fluorochrome-conjugated antibodies were as follows: anti-CD8 (53-6.7), anti-CD4 (RM4-5), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-PD-1 (J43), anti-CD45.1 (A20), anti-CD45.2 (104), anti-ICOS (C398.4A), anti-IFN-γ (XMG1.2), anti-TNFα (MP6-XT22), anti-Eomes (Dan11mag), anti-T-bet (eBio4B10), anti-IL-2 (JES6-5H4), anti-IL-7Rα (eBio17B7) and anti-KLRG1 (2F1) from eBiosciences; anti-Bcl6 (K112-91) from BD Biosciences; anti-human granzyme B (FGB12) and corresponding isotype control from Invitrogen/Life Technologies, anti-SLAM (TC15-12F12.2) from BioLegend. For detection of CXCR5, three-step staining protocol was used with unconjugated anti-CXCR5 (2G8; BD Biosciences) (24 (link)). For detection of Bcl6, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences), followed by incubation with a fluorochrome-conjugated antibody or isotype control. Peptide-stimulated cytokine production and detection by intracellular staining were as described (25 (link)). Data were collected on a FACSVerse (BD Biosciences) and were analyzed with FlowJo software (TreeStar).

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Gullicksrud J.A., Li F., Xing S., Zeng Z., Peng W., Badovinac V.P., Harty J.T, & Xue H.H. (2017). Differential requirements for Tcf1 long isoforms in CD8+ and CD4+ T cell responses to acute viral infection. Journal of immunology (Baltimore, Md. : 1950), 199(3), 911-919.

Publication 2017

anti-Bcl6 (K112-91) anti-CXCR5 (2G8; Foxp3/Transcription Factor Staining Buffer Set FACSVerse

Anti t Antibodies Antibody Bcl6 Blood Buffer Cd44 Cd62l Cells Cxcr5 Cytokine Fluorochrome Granzyme b Human Icos Ifn γ Intracellular Isotype Klrg1 Lymph nodes Lymphocytes Peptide Spleen Tnfα Transcription factor

Corresponding Organization : University of Iowa

Other organizations : George Washington University

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Variable analysis

independent variables

Single-cell suspensions prepared from the spleen, lymph nodes (LNs), or peripheral blood lymphocytes (PBLs)

dependent variables

Surface or intracellular staining of cells
Cytokine production and detection by intracellular staining

control variables

Fluorochrome-conjugated antibodies used for staining, including anti-CD8, anti-CD4, anti-CD44, anti-CD62L, anti-PD-1, anti-CD45.1, anti-CD45.2, anti-ICOS, anti-IFN-γ, anti-TNFα, anti-Eomes, anti-T-bet, anti-IL-2, anti-IL-7Rα, and anti-KLRG1
Three-step staining protocol used for detection of CXCR5
Fixation and permeabilization of surface-stained cells for detection of Bcl6
Peptide stimulation for cytokine production and detection

positive controls

Anti-human granzyme B antibody and corresponding isotype control

negative controls

Isotype control for Bcl6 staining

Annotations

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