Flow Cytometric Analysis of Inflammatory Cells

Flow cytometry was performed essentially as previously described [16 (link),19 (link)] using FACSVerse (BD Biosciences) and data analyzed using the FACSuite software. TNF⁺ microglia (CD11b⁺CD45^dim), TNF⁺ macrophages (CD11b⁺CD45^highGr1⁻) and TNF⁺ granulocytes (CD11b⁺CD45^highGr1⁺) were identified as previously detailed [16 (link),19 (link)]. Control mice and mice allowed to survive for six and 24 hours after pMCAO were treated intravenously with either saline, XPro1595 or etanercept 30 minutes after surgery.
Prior to fixation, cells were stained for live/dead cells for 30 minutes at 4°C using a Fixable Viability Dye eFluoro 506 (eBioscience, Hatfield, UK) diluted in PBS. A total of 1,000,000 events were collected using forward scatter (FSC) and side scatter (SSC) and analysis of the live/dead gate revealed comparable numbers of dead cells in all the samples. In addition, blood and spleen samples were collected and analyzed for CD45, CD11b, Gr1, and CD3 expression.
Positive staining for TNF (Biolegend, Copenhagen, Denmark), CD11b, CD45, Gr1 and CD3 (BD Pharmingen, Albertslund, Denmark) was determined based on fluorescence levels of the respective isotype controls (Biolegend and BD Pharmingen). The mean fluorescence intensity (MFI) was calculated as the geometric mean of each population in the TNF, CD45 and CD11b positive gates, respectively.

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Clausen B.H., Degn M., Martin N.A., Couch Y., Karimi L., Ormhøj M., Mortensen M.L., Gredal H.B., Gardiner C., Sargent I.I., Szymkowski D.E., Petit G.H., Deierborg T., Finsen B., Anthony D.C, & Lambertsen K.L. (2014). Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia. Journal of Neuroinflammation, 11, 203.

Publication 2014

FACSVerse TNF

Blood Cd11b Cells Etanercept Flow cytometry Fluorescence Granulocytes Isotype Macrophages Mice Microglia Saline Spleen Surgery Xpro1595

Corresponding Organization :

Other organizations : University of Southern Denmark, Glostrup Hospital, University of Oxford, KLA (United States), Xencor (United States), Lund University

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Protocol cited in 6 other protocols

Variable analysis

independent variables

Treatment with saline
Treatment with XPro1595
Treatment with etanercept

dependent variables

Percentage of TNF+ microglia (CD11b+CD45dim)
Percentage of TNF+ macrophages (CD11b+CD45highGr1-)
Percentage of TNF+ granulocytes (CD11b+CD45highGr1+)
Mean fluorescence intensity (MFI) of TNF, CD45, and CD11b in the respective positive gates

control variables

FACSVerse (BD Biosciences) for flow cytometry
FACSuite software for data analysis
Staining protocol for live/dead cells using Fixable Viability Dye eFluoro 506
Collection of 1,000,000 events using forward scatter (FSC) and side scatter (SSC)
Analysis of the live/dead gate to ensure comparable numbers of dead cells in all samples
Collection and analysis of blood and spleen samples for CD45, CD11b, Gr1, and CD3 expression

controls

Positive controls: Isotype controls for TNF, CD45, CD11b, Gr1, and CD3 staining
Negative control: Control mice (no pMCAO surgery)

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