Anti cd63 clone tea3 18

We performed tests on the endo-lysosomal network by CD63 and CD107a staining, using anti-CD63 and anti-CD107a antibodies which are respectively endosome and lysosome markers [77 (link)]. Using flow cytometry, we can measure the intracellular content of these two molecules with previous cellular fixation and permeabilization (FIX & PERM^® cell fixation and cell permeabilization kit—Invitrogen) [78 (link)]. Niemann-Pick and wild-type B lymphocytes were washed in PBS, resuspended in 250μl of FIX reagent and incubated at 4°C for 30 min. Then the cells were washed in the perm/washing buffer and resuspended in 250μl of PERM reagent. Mouse monoclonal anti-Human Antibodies anti-CD63 (clone TEA3/18, Immunostep) and anti-CD107a (clone H4A3, BioLegend), FITC- and PerCP/Cy5.5-conjugated respectively, were added to the bottom of the tube and incubated at 4°C for 30 min, at concentrations indicated in the manufacturer’s instructions. After a washing step, the supernatant was discarded and samples were acquired by flow cytometry, collecting at least 10,000 events for each tube.

EVs were incubated with 6000 antibody-coated beads in 100 μl of PBS containing 1% casein for 18 h, in a 5 ml tube without agitation at room temperature (RT). After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated. After antibody binding, beads were washed with filtered PBS containing 1% BSA, and recovered using a Magnetic Rack (MagneSphere(R) Mag. Sep. Stand 12-hole, 12 × 75 mm (Promega, Ref Z5343). When using biotinylated antibody, a step incubating with streptavidin-PE (Immunostep, S.L.) was added followed by bead washing with PBS-1% BSA. When analysing urine samples, 500 μl of urine supernatant (see below) were directly incubated with 3000 microbeads. Samples were analyzed using either Gallios or Cytomics FC 500 (Beckman Coulter) or FACSCalibur Flow Cytometers (Becton Dickinson) and data were analysed using Kaluza (Beckman Coulter) and FlowJo (Tree Star, Inc). Single beads were gated in Forward Scatter in the region corresponding to 6 µm (established using calibration beads (FlowCheck Pro^TM fluorospheres, Beckman Coulter), excluding bead doublets and non-bead events in FSC/SSC and selecting beads using the corresponding channel of fluorescence. The percentage of single beads was above 95%.