Immunofluorescence analysis of CD63 was performed using 70% confluent log phase Jurkat cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with CD63 Recombinant Rabbit Monoclonal Antibody (142) (Product # MA5-30187) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (1:2500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytosolic punctate localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60 magnification.

Western blot was performed using Anti-CD63 Recombinant Rabbit Monoclonal Antibody (142) (Product # MA5-30187) and a 63 kDa band corresponding to CD63 was observed across cell lines tested. Purified exosome extracts (7 µg lysate) of Jurkat (Lane 1), MOLT-4 (Lane 2), Raji (Lane 3), Daudi (Lane 4) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).