Western blotting assay was conducted as previously reported [32 (link)]. The following antibodies were used: Alix (12422-1-AP, Proteintech, Wuhan, China), TSG101 (67381-1-Ig, Proteintech), CD81 (66866-1-Ig, Proteintech), Calnexin (ab133615, Abcam, Cambridge, MA, USA), iNOS (18985-1-AP, Proteintech), ARG1 (DF6657, Affinity, Changzhou, China), PD-L1 (DF6526, Affinity), PD-L1 (66248-1-IG, Proteintech), CXCL1 (AF5403, Affinity), EED (85,322 T, CST, MA, USA), and β-actin (4970S, CST).
Alix 12422 1 ap
Manufactured by Proteintech
Sourced in United States
Alix (12422-1-AP) is a lab equipment product offered by Proteintech. It serves as a core component in various protein analysis and manipulation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Cd63 25682 1 ap, by Proteintech (2 mentions)
Cd9 20597 1 ap, by Proteintech (2 mentions)
Ht7700, by Hitachi (2 mentions)
Dynapro nanostar, by Wyatt Technology (2 mentions)
Cxcl1, by Affinity Biosciences (1 mentions)
Cd81 66866 1 ig, by Proteintech (1 mentions)
Pd l1 66248 1 ig, by Proteintech (1 mentions)
Pd l1, by Affinity Biosciences (1 mentions)
Df6526, by Affinity Biosciences (1 mentions)
Calnexin, by Abcam (1 mentions)
3 protocols using alix 12422 1 ap
1
Comprehensive Protein Analysis of Extracellular Vesicles
2
Isolation and Characterization of Exosomes
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Exosomes were isolated by ultracentrifugation. Brie y, the conditioned supernatants of MSCs pretreated with or without TSA were collected and centrifuged at 300 ×g for 10 min. The supernatant was centrifuged at 10000 ×g for 30 min and ltered through a sterile 0.22-μm pore lter. The ltered solution was ultracentrifuged at 120,000 ×g for 2 h. Lastly, the supernatant was discarded to obtain the exosome precipitate [25, 26] . The structure of exosomes was observed by transmission electron microscopy (TEM) analysis using a Hitachi HT7700 (Hitachi, Japan) and their size distribution was determined by dynamic light scattering (DLS) using a DynaPro NanoStar (WYATT, USA). The exosomes were identi ed using western blotting with the marker proteins CD9 (20597-1-AP; Proteintech, Chicago, USA), CD63 (25682-1-AP; Proteintech), and Alix (12422-1-AP; Proteintech). The protein concentration in exosomes was determined using the bicinchoninic acid kit (Thermo Fisher Scienti c, Waltham, MA, USA).
3
Isolation and Characterization of Exosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Exosomes were isolated by ultracentrifugation. Brie y, the conditioned supernatants of MSCs pretreated with or without TSA were collected and centrifuged at 300 ×g for 10 min. The supernatant was centrifuged at 10000 ×g for 30 min and ltered through a sterile 0.22-μm pore lter. The ltered solution was ultracentrifuged at 120,000 ×g for 2 h. Lastly, the supernatant was discarded to obtain the exosome precipitate [25, 26] . The structure of exosomes was observed by transmission electron microscopy (TEM) analysis using a Hitachi HT7700 (Hitachi, Japan) and their size distribution was determined by dynamic light scattering (DLS) using a DynaPro NanoStar (WYATT, USA). The exosomes were identi ed using western blotting with the marker proteins CD9 (20597-1-AP; Proteintech, Chicago, USA), CD63 (25682-1-AP; Proteintech), and Alix (12422-1-AP; Proteintech). The protein concentration in exosomes was determined using the bicinchoninic acid kit (Thermo Fisher Scienti c, Waltham, MA, USA).
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