外泌体示踪

denisqiang

同求，谢谢

denisqiang

Isolation and labeling of exosomes
B16-F10 melanoma exosomes were isolated for use in experiments according to previously established methods (15). Briefly, B16-F10 melanoma cell cultures were grown to 70% confluence in three 300 cm2 flask. Culture media was removed and cells washed in PBS. Cells were cultured for 48 hours in the presence of conditioned media. Conditioned culture media was prepared by subjecting normal culture media to overnight ultracentrifugation at 110,000 × g to remove bovine exosomes (19). B16 melanoma exosomes were collected from 48-hour culture in conditioned media through standard differential centrifugation steps using a 70 Ti rotor (19). Culture media was spun and supernatants collected from 300 × g for 10 minutes, 2,000 × g for 10 minutes, to remove residual cells and debris, 10,000 × g for 30 minutes to remove microparticles (20) and 100,000 × g for 2 hours in the presence or absence of 1.0 μmol/L DiI or DiR. Exosome pellets were washed 3 times in PBS, pooled, and reisolated in PBS at 100,000 × g for 2 hours. Exosome pellets were resuspended in 1 mL of PBS, protein content measured via BCA absorbance (Thermo Fisher Scientific Inc.) and stored at −80°C until use. Between the 10,000 and 100,000 × g centrifugation steps, exosomes were sized using dynamic light scattering (DLS) as previously reported (15) and the electrokinetic potential (zeta potential) of purified exosomes in PBS was measured using a Zeta Plus Zeta Potential Analyzer (Brookhaven Instruments Corp.). Previously, fluorescent exosome localization technique (FELT) was used to confirm the use of differential centrifugation and DLS in obtaining a purified population of B16-F10 melanoma exosomes from conditioned media (15). Using FELT, B16-F10 melanoma exosomes applied to continuous sucrose gradients (2.0–0.25 mol/L sucrose, 20 mmol/L HEPES/NaOH, pH 7.4), were found to have a density of (1.10–1.21 g/mL; ref. 15).

denisqiang

View captured exosomes on magnetic beads
https://www.systembio.com/exo-glow
Exosomes from HEK293 cells grown in Exo-FBS exosome-depleted media supplement with standard DMEM were isolated using ExoQuick-TC. The exosome pellet was resuspended in 1 ml 1x PBS and contained an exosome protein content of 1 ug/ul. About 500 ul of this exosome suspension was labeled with 50 ul of either 10x Exo-Red or Exo-Green for 10 minutes at 37°C. The exosomes were re-isolated using the addition of 100 ul ExoQuick-TC (included in kits) and precipitation at 5°C for 30 minutes on ice or 4°C. The labeled exosome pellets were resuspended in 500 ul 1x PBS. CD63-coupled magnetic beads from SBI’s Exo-Flow IP kit (cat# EXOFLOW32A-CD63). Briefly, 50 ul of CD63 magnetic beads were incubated with 100 ul of the labeled exosomes overnight at 4°C on a rotator. The following day, the bead/labeled exosomes were placed on a magnetic plate for 5 minutes and then washed with 100 ul 1x wash buffer once. Then, 100 ul 1x PBS was added to the IP well and placed on the magnetic rack for 2 minutes to position the beads at the bottom of the wells. The beads with captured, labeled exosomes were imaged using fluorescent microscopy.

denisqiang

http://aatbio.com/protocol/22033.pdf

好了，有这3个，基本全了