Flow Cytometry Analysis of Immune Cell Populations

All infected and uninfected tissues were dissociated by collagenase digestion as described previously ^{40 (link)}. Cell suspensions were stained and analyzed by flow cytometry. The following antibodies were used for cell surface staining: anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD3 (145-2C11), anti-Ly6C (HK1.4), anti-MHCII (M5/114.15.2), anti-CD19 (ebio1D3), anti-NK1.1 (PK136), anti-siglecH (ebio440c), anti-B220 (RA3-6B2), anti-CD4 (GK.5), anti-CD8 (53-6.7) and anti-CD69 (H1.2F3) were purchased from eBioscience, anti-Ly6G (1A8) was obtained from BD Pharmingen, anti-Ly6-B2 antibody (7/4) from AbD Serotec, and CCR2 (475301) from R&D Systems. For intracellular staining: anti-iNOS antibody was purchased from Millipore, and anti-TNF-α (MP6-XT22) was purchased from BD Pharmingen. Cell suspensions from collagenase-digested tissue were stained for cell surface markers and permeabilized using the BD Cytofix/Cytoperm Kit (BD Pharmingen). For detection of intracellular TNF-α, cells were also treated ex vivo with GolgiPlug (BD Pharmingen) for 6 h at 37°C in complete RPMI medium prior to cell surface staining and stained according to the manufacturers instructions. Acquisitions were performed with a BD LSRII, and data were analyzed with the FlowJo software.

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Han S.J., Melichar H.J., Coombes J.L., Chan S.W., Koshy A.A., Boothroyd J.C., Barton G.M, & Robey E.A. (2014). Internalization and TLR-dependent type I interferon production by Inflammatory monocytes to Toxoplasma gondii. Immunology and cell biology, 92(10), 872-881.

Publication 2014

anti-CD8 (53-6.7) anti-CD69 (H1.2F3) anti-Ly6G (1A8) anti-Ly6-B2 antibody (7/4) CCR2 (475301) anti-iNOS antibody anti-TNF-α (MP6-XT22) BD Cytofix/Cytoperm Kit GolgiPlug BD LSRII

Anti antibody Anti cd3 Antibodies Cd11b Cell Clone Collagenase Digestion Flow cytometry Inos Intracellular Tissue Tnf α

Corresponding Organization :

Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, University of California, Berkeley, University of Liverpool, University of Arizona, Stanford University

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Variable analysis

independent variables

Collagenase digestion to dissociate infected and uninfected tissues

dependent variables

Cell surface marker expression (CD11b, CD11c, CD3, Ly6C, MHCII, CD19, NK1.1, siglecH, B220, CD4, CD8, CD69, Ly6G, Ly6-B2, CCR2)
Intracellular marker expression (iNOS, TNF-α)

control variables

Positive control: Not specified
Negative control: Not specified

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