Twelve-parameter Flow Cytometry Analysis

Twelve-parameter flow cytometric analysis was performed on WB, LN, RB and BM derived cells according to standard procedures using a panel of monoclonal antibodies that we and others have shown to be cross-reactive with RM [35] (link)–[37] (link). Predetermined optimal concentrations were used of the following antibodies: anti-CD3-Alexa700 (clone SP34-2), anti-CD3-APC-Cy7 (clone SP34-2), anti-CD4-Pacific Blue (clone OKT4), anti-CD8-APC-Cy7 (clone SK1), anti-CD95-PE-Cy5 (clone DX2), anti-CCR5-APC (clone 3A9), anti-Ki-67-Alexa700 (clone B56), anti-Ki-67-FITC (clone B56), anti-CD14-Pe-Cy7 (clone M5E2), anti-CD16-BV421 (clone 3G8), anti-CD62L-PE (clone SK11), anti-CCR7-PE-Cy7 (Clone 3D12) (all from BD Pharmingen); anti-CD28-ECD (clone CD28.2) (Beckman Coulter); anti-CD8-Qdot705 (clone 3B5) and Aqua Live/Dead amine dye-AmCyan (Invitrogen). Intracellular staining for Ki-67 was performed at room temperature for 30 minutes following permeabilization with cytofix/cytoperm (BD Bioscience). Flow cytometric acquisition was performed on an LSRII cytometer driven by the FACS DiVa software. Analysis of the acquired data was performed using FlowJo software (TreeStar) and graphs were prepared using Prism version 6.0 (GraphPad).

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Micci L., Alvarez X., Iriele R.I., Ortiz A.M., Ryan E.S., McGary C.S., Deleage C., McAtee B.B., He T., Apetrei C., Easley K., Pahwa S., Collman R.G., Derdeyn C.A., Davenport M.P., Estes J.D., Silvestri G., Lackner A.A, & Paiardini M. (2014). CD4 Depletion in SIV-Infected Macaques Results in Macrophage and Microglia Infection with Rapid Turnover of Infected Cells. PLoS Pathogens, 10(10), e1004467.

Publication 2014

anti-CD28-ECD (clone CD28.2) anti-CD8-Qdot705 (clone 3B5) Aqua Live/Dead amine dye-AmCyan cytofix/cytoperm Prism version 6.0

Amine Anti cd3 Antibodies Ccr5 Cd62l Cells Clone Cross reactive Fitc Flow cytometric Intracellular Monoclonal antibodies Prism

Corresponding Organization : Emory University

Other organizations : Tulane University, Leidos (United States), Frederick National Laboratory for Cancer Research, University of Pittsburgh, Georgia Department of Public Health, University of Miami, University of Pennsylvania, UNSW Sydney

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Variable analysis

independent variables

Cell types (WB, LN, RB, BM)

dependent variables

Expression of cell surface markers (CD3, CD4, CD8, CD95, CCR5, CD14, CD16, CD62L, CCR7, CD28)
Expression of intracellular marker (Ki-67)

control variables

Predetermined optimal concentrations of monoclonal antibodies
Permeabilization procedure for intracellular staining
Flow cytometric acquisition on an LSRII cytometer

controls

Positive controls: The authors mention that the panel of monoclonal antibodies used has been shown to be cross-reactive with RM (Rhesus Macaque) in previous studies [35-37].
Negative controls: No explicit negative controls are mentioned in the provided information.

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