2.2 Blood samples
Venous blood was collected from each of the PNS patients and each of the
healthy controls and stored in ethylenediamine tetraacetic acid
(EDTA)-Na2 as an anti-coagulant. Peripheral blood
mononuclear cells (PBMCs) were isolated by Ficoll density gradient for
subsequent flow cytometric analysis . Plasma was obtained after
centrifugation and stored at -80°C for analysis by cytometric bead array
(CBA). CD4+CD25- T cells were
immediately isolated from the patients’ peripheral blood samples,
according to the manufacturer’s instructions for microbeads (11363D,
Dynal; Invitrogen, USA). Cell
populations were considered pure at > 97% by flow
cytometry, and cell activity was investigated using the trypan blue
exclusion assay (using the threshold of > 95% of the cell
population as a positive indicator of significantly decreased cell
activity).