Evaluating Endolysosomal Network by Flow Cytometry

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.