Analyzing Human Regulatory T Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from 20 to 25 ml of peripheral blood samples via Ficoll density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). According to the recommendation of the international working group of the experts in flow cytometry [7 (link)], CD3, CD4, CD25, CD127, and FoxP3 markers were used to analyze human Treg cells using a gating strategy illustrated in Supplementary Figure 1. Specifically, CD3-FITC, CD45-PerCP, CD127-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-Alexa Fluor® 700, (EXBIO a.s., Prague, Czech Republic), Foxp3-PE, and CD25-Brilliant Violet (BioLegend, San Diego, USA) antibodies (Abs) were used. For intracellular staining, we used Foxp3-Fix/Perm and Foxp3-PErm Buffers (BioLegend, San Diego, USA). The flow cytometric data were collected on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Summit 4.3 software (Dako, Glostrup, Denmark).

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Stechova K., Sklenarova-Labikova J., Kratzerova T., Pithova P, & Filipp D. (2017). Not Only Glycaemic But Also Other Metabolic Factors Affect T Regulatory Cell Counts and Proinflammatory Cytokine Levels in Women with Type 1 Diabetes. Journal of Diabetes Research, 2017, 5463273.

Publication 2017

Ficoll density gradient centrifugation CD3-FITC CD45-PerCP CD127-APC Foxp3-PE Foxp3-Fix/Perm LSR II flow cytometer Summit 4.3 software

Antibodies Blood Buffers Cd25 Density gradient centrifugation Ficoll Fitc Flow cytometric Human Intracellular Peripheral blood mononuclear cells Perm Treg cells Violet

Corresponding Organization : Charles University

Other organizations : Czech Academy of Sciences, Institute of Molecular Genetics

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Variable analysis

independent variables

Peripheral blood mononuclear cells (PBMCs) isolation via Ficoll density gradient centrifugation

dependent variables

Analysis of human Treg cells using CD3, CD4, CD25, CD127, and FoxP3 markers

control variables

Peripheral blood samples (20 to 25 ml)
Antibodies: CD3-FITC, CD45-PerCP, CD127-APC, CD4-Alexa Fluor® 700, Foxp3-PE, and CD25-Brilliant Violet
Foxp3-Fix/Perm and Foxp3-Perm Buffers for intracellular staining
Flow cytometric data collected on LSR II flow cytometer and analyzed using Summit 4.3 software

controls

No positive or negative controls explicitly mentioned

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