Mce filter

A total of 36 HAM/TSP, 10 ND, and 2 HTLV-II patient PBMC samples were used for ex vivo incubation and later exosome isolation. PBMCs were isolated by Ficoll-Hypaque (Lonza, Walkersville, MD) centrifugation, and were cryopreserved in liquid nitrogen prior to use. All NDs were noted to be healthy and HTLV-1 negative. Briefly, PBMCs were placed in Exo-free CRPMI at 8 × 10⁶ cells per well in a 12-well plate and incubated at 37 °C for 5 days. ND PBMCs were maintained in culture with the addition of 100 IU/mL recombinant human (rh) IL-2. Culture supernatant was collected and then spun at 1300 rpm for 10 min to remove cells. Spun supernatant was then pushed through a 0.22 μm MCE filter (EMD Millipore) to remove large debris, apoptotic bodies and extracellular vesicles > 220 nm. Cerebrospinal fluid (CSF) was obtained through lumbar puncture of study participants by neurologist and nurse practitioners of the Neuroimmunology Clinal group. CSF was spun at 1300 rpm for 10 min to remove cells and supernatant was stored in 1 mL aliquots at − 80 °C prior to use.

The in vitro drug release from tablets (n = 6) was determined using a type II dissolution apparatus (AT7, Sotax, Aesch, Switzerland) with a paddle speed of 100 rpm. The experiments were performed in 900 mL 0.1 N HCl solution at 37.0 ± 0.5 °C. Samples were withdrawn at pre-set times (between 0 and 180 min), filtered through a 0.22 μm MCE filter (Merck, Burlington, MA, USA) and analyzed spectrophotometrically at 240 nm (U-1900 UV−VIS Spectrophotometer, Hitachi, Tokyo, Japan). Fresh dissolution medium was added after each sample was collected to keep the volume inside the vessel constant.