Mab81751

The following primary antibodies were used in Western blotting experiments: anti-MTf (R&D systems, MAB8175), CD63 (Abcam, ab134045), Tsg101 (Abcam, ab83), Alix (Abcam, ab186429), Flotillin-1 (BD Biosciences, clone 18), hsc70 (Stressgen, 1B5), CD9 (GeneTex, GTX62294), Lyn (Santa Cruz, sc-7274), syntenin-1 (GeneTex, GTX108470), CEACAM6 (Temecula, MABT 323). HRP-conjugated secondary antibodies used for Western blot and ELISA were purchased from Jackson ImmunoResearch Laboratories.
Mouse monoclonal anti-MTf (R&D Systems, MAB81751, dilution 1/50) antibodies were used for flow cytometry and immunofluorescence using anti-mouse IgG conjugated with Alexa Fluor 488 (Invitrogen, A11029, dilution 1/2000) as secondary antibodies. Cholera toxin B-chain conjugated with Alexa Fluor 555 was from Thermo Fischer scientific (C-34775).
Anti-CD63 (Abcam, ab8219) was used for exosome immunoisolation. Anti-CD63 (Abcam, ab252919) and anti-MTf (R&D systems, MAB81751 or Abcam, ab201922) antibodies were used for immunocapture-based ELISA.

Wells of a 96-well plate (Nunc Maxisorb) were coated with 100 ng of anti-CD63 antibody (ab252919, Abcam) in 100 µl carbonate buffer (pH 9.6) overnight at 4°C with gentle agitation, washed twice with PBS and blocked by PBS-5% BSA for 2 h at room temperature (RT). Plates were then washed three times with PBS, and 100 µl volume of exosomes -or plasma diluted 1/20 in PBS -were incubated for 2 h at RT. After five washes with PBS, the wells were added with anti-MTf antibody (50 ng of MAB81751, R&D Systems) in PBS-5% BSA for 2 h at RT, washed again five times with PBS, and antimouse IgG-HRP (Jackson Immunoresearch) -diluted (1/5000) in PBS-5% BSA -was incubated for 1h at RT. After PBS washes, 100 µl TMB solution (UP664781) was added to the wells, color development was stopped by adding 50 µl H 2 SO 4 (1 M) after 10-20 min incubation and absorbance was recorded at . CC-BY-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 9, 2020. ; https://doi.org/10.1101/2020.11.09.373852 doi: bioRxiv preprint 450 nm using a TECAN Sunrise spectrophotometer. When indicated, exosomes were pre-incubated for 30 min at RT with 1% Triton X-100 or PI-PLC from Bacillus cereus (0.5 u).