Fig. 8. Quantitation of exosome release by western blot and by measuring exosome-associated acetyl-cholinesterase and N-Rh-PE. (A) Samples of the exosomal fraction (see Materials and Methods) were solubilized in reducing SDS loading buffer, boiled and subjected to SDS-PAGE. The proteins were transferred to nitrocellulose membranes and detected with antibodies against the transferrin receptor (TfR), the protein Lyn and the heat shock protein Hsc70, respectively. The corresponding bands were detected using an enhanced chemiluminescence detection kit. A shows representative western blots of at least three independent experiments. Densitometric quantitation of the bands is depicted in B. Data are the mean±s.e.m. of three experiments expressed as a percentage of the control cells transfected with the vector alone. ^*P<0.05. (C) An aliquot of the exosome fraction (see Materials and Methods) was suspended in 100 µl phosphate buffer and incubated with 1.25 mM acetylthiocholine and 0.1 mM 5,5'-dithio-bis(2-nitrobenzoic acid) in a final volume of 1 ml. The incubation was carried out in cuvettes at 37°C, and the change in absorbance at 412 nm was followed continuously for 30 minutes. Data were normalized as the percentage of the maximal activity measured at 30 minutes in the vector-transfected cells. Data represent the mean±s.e.m. of four independent experiments. (D) Cells were labeled for 60 minutes at 4°C with the fluorescent lipid analog N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) as described in the Materials and Methods. Cells were extensively washed with cold PBS to remove excess unbound lipid and were then cultured in complete RPMI medium to collect exosomes. A sample of the exosomal fraction was suspended in 1.5 ml PBS-TX100 (0.1%) and N-Rh-PE was measured by fluorometry. Data represent the mean±s.e.m. of three independent experiments expressed as a percentage of the control (vector transfected cells). Statistical analysis: t-test for single group mean. ^*Mean differs from control P<0.05; ^**mean differs from control P<0.01.