Flow Cytometry Analysis of Regulatory T-Cells

Staining was performed on peritoneal exudate cells using the following anti-mouse antibodies and flurorochrome conjugates: anti-CD4 with Biotin (Miltenyi Biotec), anti-CD25 with PE (eBioscience) and anti-Foxp3 with APC (Miltenyi Biotec). Secondary staining for biotinylated CD4 was done with either FITC- or Pe-Cy5-conjugated streptavidin (BD Biosciences). As isotype control of activated/regulatory T-cells, mouse IgG1 K isotype (APC, Miltenyi Biotec) and Rat IgG1 K Isotype (PE, eBioscience) were used. The staining procedure was executed as previously described (6 (link)) and results acquired on a cytometer (FACSCalibur^TM, Beckton Dickinson) and analyzed on FlowJo software (Tree Star, United States).

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Nono J.K., Lutz M.B, & Brehm K. (2020). Expansion of Host Regulatory T Cells by Secreted Products of the Tapeworm Echinococcus multilocularis. Frontiers in Immunology, 11, 798.

Publication 2020

Pe-Cy5-conjugated streptavidin FACSCaliburTM

Anti antibodies Biotin Cd25 Cells Fitc Igg1 Isotype Mouse Peritoneal exudate Regulatory t cells Streptavidin Tree

Corresponding Organization : University of Würzburg

Other organizations : University of Cape Town, Ministry of Scientific Research and Innovation

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Variable analysis

independent variables

Anti-CD4 with Biotin (Miltenyi Biotec)
Anti-CD25 with PE (eBioscience)
Anti-Foxp3 with APC (Miltenyi Biotec)
FITC- or Pe-Cy5-conjugated streptavidin (BD Biosciences)

dependent variables

Peritoneal exudate cells

control variables

Staining procedure as previously described (6 (link))
FACSCalibur^TM cytometer (Beckton Dickinson)
FlowJo software (Tree Star, United States)

positive controls

Mouse IgG1 K isotype (APC, Miltenyi Biotec)
Rat IgG1 K Isotype (PE, eBioscience)

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