Comprehensive Immunophenotyping of PBMCs

0.5 x 10⁶ isolated PBMCs were stained with anti-CD3-PacBlue, CD4-APC-Alexa Fluor750, CD8-PerCP-CY5.5, CD27-APC, CD45RO-PE, CD57-FITC, and CD38-PE-Cy7 (eBioscience) to distinguish T cell subsets and levels of activation. B cell subsets and activation status was analyzed by staining with CD70-FITC, α-IgD-PE (BD), CD19-PE-Cy7, and CD27-APC (BD) antibodies. Appropriate isotype controls were used to determine the percentages of cells expressing the respective markers (Biolegend, San Diego, CA). Samples were analyzed on LSR-II flow cytometer (BD) and data analysis was performed using the FlowJo (FlowJo, LLC) and FACSDiva software (BD) as described [52 (link), 54 (link), 55 (link)].

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Hel Z., Xu J., Denning W.L., Helton E.S., Huijbregts R.P., Heath S.L., Overton E.T., Christmann B.S., Elson C.O., Goepfert P.A, & Mestecky J. (2017). Dysregulation of Systemic and Mucosal Humoral Responses to Microbial and Food Antigens as a Factor Contributing to Microbial Translocation and Chronic Inflammation in HIV-1 Infection. PLoS Pathogens, 13(1), e1006087.

Publication 2017

CD8-PerCP-CY5.5 CD27-APC CD45RO-PE CD57-FITC CD38-PE-Cy7 CD19-PE-Cy7 LSR-II flow cytometer FACSDiva software

Anti cd3 Antibodies Cd45ro Cy5 5 Fitc Isotype T cell subsets

Corresponding Organization : Charles University

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Variable analysis

independent variables

Staining with anti-CD3-PacBlue, CD4-APC-Alexa Fluor750, CD8-PerCP-CY5.5, CD27-APC, CD45RO-PE, CD57-FITC, and CD38-PE-Cy7 antibodies
Staining with CD70-FITC, α-IgD-PE, CD19-PE-Cy7, and CD27-APC antibodies

dependent variables

Percentages of T cell subsets and levels of activation
B cell subsets and activation status

control variables

Appropriate isotype controls used to determine the percentages of cells expressing the respective markers

controls

Positive controls: Not specified
Negative controls: Isotype controls

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