Lymphosep

A gradient centrifugation method over the LymphoSep (Biosera, UK) was employed to isolate PBMCs from the heparinized peripheral blood. The PBMCs layer was carefully harvested and washed three times with Roswell Park Memorial Institute (RPMI)-1640 medium. Then, the PBMCs were again suspended in the supplemented-RPMI-1640 medium (a medium contained 10% heat inactivated fetal bovine serum [Gibco Life Technologies Ltd, Paisley, UK], 100 U/mL of penicillin, and 100 μg/mL of streptomycin).
The PBMCs were then dispensed in the 24-well sterile flat-bottomed microtiter plates (1×10⁶ cell/well) and cultured (at 37°C in a 5% CO2 incubator for 24 hours) in the absence of a stimulator, in the presence of MOG (35–55) human (Anaspec, USA) or PHA (Gibco Life Technologies Ltd, Paisley, UK) at a concentration of 10 μg/mL. After this time, the total RNA was extracted from the PBMCs for more analyses.

Ten ml of venous blood were collected in vials containing ethylenediaminetetraacetic acid (EDTA). This was performed at the beginning of therapy (day 1 of RT) and one week after the last fraction of RT.
Plasma and peripheral blood mononuclear cells (PBMCs) were isolated after density gradient centrifugation using a 1.077 g/mL synthetic epichlorohydrin sucrose polymer Lymphosep [Lymphocyte Separation Media-500 mL; density 1077 g/mL; Cat no: LM-T1702/500; Biosera, Cholet, France], at a 1:1 ratio of the total blood volume. Centrifugation of blood samples was performed at 1500 rpm (30 min, 25 °C). The plasma layer was transferred in sterile Eppendorf tubes and was gradually frozen at −20 °C and subsequently at −80 °C. The PBMC layer was also collected for further studies and stored at −20 °C.

PBMCs were isolated using lymphosep (density 1077 g/mL, Biosera, Nuaille, France) from freshly collected blood; blood was then diluted (1:1 dilution) with phosphate-buffered saline (PBS). The diluted whole blood was carefully layered over the separation medium (1/2 × the volume of the sample), and the two phases were kept separated before the centrifugation. Samples were then centrifuged at 400× g for 30 min at 20 °C. PBMCs were collected after aspiration of the plasma and platelets layer; cells were then washed with PBS and processed for proteasome peptidase activity [6 (link)].