Exosome Isolation and Purification Kit (from cell culture media)

1. Pretreatment of cell culture medium

1. Collect cell culture supernatant and centrifuge at 3,000 × g for 10 min at 4°C to remove cells and large debris.

2. Transfer the supernatant to a new tube, centrifuge at 10,000 × g for 10 min at 4°C, and collect the clarified supernatant.

2. Exosome enrichment

1. Add 1 mL Exosome Concentration Solution (ECS) per 4 mL clarified supernatant. Thoroughly mix Exosome Concentration Solution (ECS) before use.

2. Vortex for 1 min and incubate at 4°C for at least 2 h. Longer incubation may increase yield, but incubation longer than 24 h is not recommended.

3. Centrifuge at 10,000 × g for 60 min at 4°C and carefully discard the supernatant. The exosome pellet may not be visible.

4. Thoroughly resuspend the exosome pellet in an appropriate amount of pre-cooled 1× PBS.

5. Centrifuge at 12,000 × g for 2 min at 4°C and collect the supernatant as crude exosomes. If debris remains, repeat the 2 min centrifugation.

3. Exosome purification and storage

1. Transfer crude exosomes to an Exosome Purification Filter (EPF), centrifuge at 3,000 × g for 10 min at 4°C, and collect the purified exosome filtrate.

2. Aliquot purified exosomes and store at -80°C. Avoid repeated freeze-thaw cycles. The purified exosomes may be used for EM, NTA, Western Blot, qPCR, and other downstream analyses.