Multiparametric Immunophenotyping of PBL

PBL subsets were determined as described previously [24 (link), 25 (link)]. For analysis of cell surface determinants, PBL were incubated with fluorochrome-labelled monoclonal antibodies against CD4 (clone RPA-T4), CD25 (clone M - A251), CD127 (clone HIL-7R-M21) (all from BD Biosciences). Intracellular determinants were stained with fluorochrome-labelled monoclonal antibodies against Foxp3 (clone 236A/E7), IFNg (clone B27), CD152 (BN13) and Helios (clone 22 F6) (all BD Biosciences). Briefly, PBL were incubated with combinations of monoclonal antibodies for 30 min and eight-color fluorescence was analyzed using a FACSCanto II triple-laser flow cytometer (BD Biosciences) [24 (link), 25 (link)]. When, in addition, intracellular proteins were studied, cell membranes were permeabilized using BD Perm/Wash buffer (BD Biosciences). At least 100,000 events were analyzed in the initial FSC/SSC dot plot.

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Aly M.G., Trojan K., Weimer R., Morath C., Opelz G., Tohamy M.A, & Daniel V. (2016). Low-dose oral cholecalciferol is associated with higher numbers of Helios+ and total Tregs than oral calcitriol in renal allograft recipients: an observational study. BMC Pharmacology & Toxicology, 17, 24.

Publication 2016

FACSCanto II triple-laser flow cytometer BD Perm/Wash buffer

Buffer Cd152 Cd25 Cell Cell membranes Clone Fluorescence Fluorochrome Ifng Intracellular Monoclonal antibodies Perm Proteins

Corresponding Organization : University Hospital Heidelberg

Other organizations : University of Giessen, Assiut University

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Variable analysis

independent variables

Monoclonal antibodies against CD4, CD25, CD127, Foxp3, IFNg, CD152, and Helios

dependent variables

Cell surface determinants
Intracellular determinants

control variables

PBL subset determination as previously described [24, 25]
Incubation of PBL with combinations of monoclonal antibodies for 30 min
Eight-color fluorescence analysis using a FACSCanto II triple-laser flow cytometer (BD Biosciences)
Permeabilization of cell membranes using BD Perm/Wash buffer (BD Biosciences) for intracellular protein staining
Analysis of at least 100,000 events in the initial FSC/SSC dot plot

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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