Confirming biological activity of ExoQuick purified exosomes

• RAW 264.7 cells were plated in a 24-well plate at a density of 5X10^5 cells/ml.
• AW 264.7 cells were left untreated (i.e. Resting cells) or treated with 10ug of exosomes* (as determined by BCA) for 24 hours.
• Supernatants from the Raw 264.7 cells were collected 24 hours after treatment.
• ELISA for measuring TNF-α was performed using eBiosciences ELISA kit.
Cells were treated with 10ug of exosomes suspended in 200ul of serum free DMEM. Cells treated with ExoQuick solution alone were treated with a volume equivalent of 200ul.

• Myco smeg: Are exosomes isolated from the culture supernatant of Raw 264.7 cells 48 hour post-infection with Mycobacterium smegmatis.
• Uninfected exosomes: Are exosomes isolated from the culture supernatant of uninfected Raw 264.7 cells.
• Crude: The pellet of culture supernatant containing exosomes but not yet purified by ExoQuick or sucrose gradient.
• Empty well: reagents only, no material from Raw 264.7 cells
• Resting cells: Raw 264.7 cells not treated with any exosomes
Data courtesy of Jeff Schorey, Ph.D., University of Notre Dame.

Bhatnagar S, Schorey JS. Exosomes released from infected macrophages contain Mycobacterium avium glycopeptidolipids and are proinflammatory. J Biol Chem. 2007 Aug 31;282(35):25779-89.