Multiparameter Flow Cytometry Assay

Flow cytometry was performed as described previously (6 (link)). Isolated cells were incubated with predetermined optimized concentrations of anti-CD3-Alexa fluor 700 (clone SP34-2, BD Pharmingen), anti-CD8-Pacific Blue or PE-CF594 (clone RPA-T8, BD Pharmingen), anti-CD20-APC-Cy7 (clone L27, BD Pharmingen), anti-CXCR5-phycoerythrin (PE) (Clone MU5UBEE, eBioscience), anti-CD200-APC (clone OX104, eBioscience), anti-CD4-PerCP (clone L200, BD Pharmingen), anti-CD95-PE-Cy5 (clone DX2, BD Pharmingen), anti-CCR7-APC-Cy7 (clone 3D12, Biolegend), anti-PD-1-PE-Cy7 (clone EH12.2H7, Biolegend), anti-ICOS-Pacific Blue (C398.4A, Biolegend) and Live/Dead dye-Alexa430 (Invitrogen). After staining the cell surface, cells were permeabilized and fixed by BD perm wash (BD Pharmingen) and washed twice. Finally, cells were incubated with anti-Ki67-FITC (B56, BD Pharmingen), washed twice and diluted in 1% PFA. Data were acquired on a LSRII flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (version 9.2 Tree Star).

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Hong J.J., Amancha P.K., Rogers K.A., Courtney C.L., Havenar-Daughton C., Crotty S., Ansari A.A, & Villinger F. (2014). Early lymphoid responses and germinal center formation correlates with lower viral load set points and better prognosis of SIV infection. Journal of immunology (Baltimore, Md. : 1950), 193(2), 797-806.

Publication 2014

anti-CD3-Alexa fluor 700 (clone SP34-2, anti-CD20-APC-Cy7 (clone L27, anti-CD4-PerCP (clone L200, anti-CD95-PE-Cy5 (clone DX2, anti-PD-1-PE-Cy7 clone EH12.2H7, BD perm wash anti-Ki67-FITC (B56, LSRII flow cytometer

Anti cd3 Cd200 Cells Clone Cxcr5 Fitc Flow cytometry Icos Perm Phycoerythrin Tree

Corresponding Organization :

Other organizations : Emory University, La Jolla Institute For Allergy & Immunology

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Variable analysis

independent variables

Anti-CD3-Alexa fluor 700 (clone SP34-2, BD Pharmingen)
Anti-CD8-Pacific Blue or PE-CF594 (clone RPA-T8, BD Pharmingen)
Anti-CD20-APC-Cy7 (clone L27, BD Pharmingen)
Anti-CXCR5-phycoerythrin (PE) (Clone MU5UBEE, eBioscience)
Anti-CD200-APC (clone OX104, eBioscience)
Anti-CD4-PerCP (clone L200, BD Pharmingen)
Anti-CD95-PE-Cy5 (clone DX2, BD Pharmingen)
Anti-CCR7-APC-Cy7 (clone 3D12, Biolegend)
Anti-PD-1-PE-Cy7 (clone EH12.2H7, Biolegend)
Anti-ICOS-Pacific Blue (C398.4A, Biolegend)

dependent variables

Measured cell surface expression of CD3, CD8, CD20, CXCR5, CD200, CD4, CD95, CCR7, PD-1, and ICOS

control variables

Live/Dead dye-Alexa430 (Invitrogen)
Cell permeabilization and fixation using BD perm wash (BD Pharmingen)

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