Isolation of migrasomes from tissue culture cells

The isolation procedure is based on a modification of previous protocols (Ma et al, 2014). Briefly, cells were grown in 150 mm dishes coated with 0.1 μg/ml fibronectin in full DMEM medium for 12 hr. A batch of purification requires 30-80 dishes. The cells and migrasomes on plates were digested with trypsin and collected in 50ml tubes. All subsequent manipulations were performed at 4°C. Cells and large debris were removed by centrifugation at 1000 g for 10 min followed by 4000 g for 20 min. Crude migrasomes were then collected as the pellet by centrifugation at 20,000 g for 30 min. The crude migrasome pellets were washed with PBS and centrifuged down again at 20,000 g for 30 min. Migrasome fractionation was performed by density gradient centrifugation, using Optiprep as the density medium (Sigma-Aldrich, D1556). First, a step gradient was built starting with 30% (500 μl), followed by 25% (500 μl), crude migrasomes 19% (500 μl), 15% (500 μl), 12% (500 μl), 10% (500 μl), 8% (500 μl), 5% (500 μl) and 2% (500 μl). The crude migrasome sample was prepared by first resuspending the pellet with 137.5 μl dilution buffer and then mixing with 400 μl 1ⅹextraction buffer and 252.5 μl 60% Optiprep. Second, the prepared gradient was centrifuged at 150,000 g for 4 hr at 4°C in an MLS-50 rotor (Beckman). Third, samples were collected from top to bottom (480 μl per fraction). Each fraction was mixed with the same volume of PBS (480 μl) and centrifuged at 20,000 g for 30 min to collect the pellet. The pellets were washed with PBS and centrifuged again at 20,000 g for 30 min. The samples were compatible with western blot analysis, TEM, cryo-EM and mass spectrometry.

P.S. Dilution buffer and extraction buffer are from the Lysosome Isolation Kit (Sigma-Aldrich, LYSISO1). Optiprep is diluted by the dilution buffer.

Isolation of migrasomes from serum samples

Blood samples of normal donors were collected for research purposes in 6-ml procoagulation vacuum tubes using standard venepuncture protocols. Human serum was withdrawn from the tube for the purification procedures. A total of 50 ml of cell-free serum was required for one batch purification. Large debris was removed by centrifugation at 1,000 g for 10 min followed by 4000 g for 20 min. Crude migrasomes were collected by centrifugation at 20,000 g for 30 min, then washed with PBS. Migrasome fractionation was performed by Iodixanol-sucrose density gradient centrifugation, using Optiprep (Sigma-Aldrich, D1556) as the density medium. First, a step gradient was built starting from 30% (500 μl), followed by 25% (500 μl), 19% (500 μl), 15% (500 μl), 12% (500 μl), 10% (500 μl), 8% (500 μl), and 5% (500 μl). The sample (500 μl) was prepared in 2% Optiprep and layered on top. The gradient was centrifuged at 150,000 g for 4 hr at 4°C in an MLS-50 rotor (Beckman). Samples were collected from top to bottom (480 μl per fraction). Each fraction was mixed with the same volume of PBS (480 μl), then centrifuged at 20,000 g for 30 min. The migrasome pellets were washed with PBS and centrifuged again at 20,000 g for 30 min. The samples were compatible with western blot analysis, TEM, cryo-EM and mass spectrometry.

Reference

Supplementary data of

Zhao X, Lei Y, Zheng J, et al. Identification of markers for migrasome detection[J]. Cell discovery, 2019, 5(1): 1-4.