To measure the luciferase activity of EVs released by HEK293T cells post-transfection, EVs containing Cas9-Nluc-CRY2 protein were purified from the culture supernatants and resuspended in PBS. The purified EVs were mixed with Nano-Glo substrate diluted in buffer (Promega) and then subjected to luciferase activity detection using EnVision Multimode Plate Reader (PerkinElmer).
To detect the luciferase activity of EVs uptake by recipient cells, isolated EVs were added to Huh7 cells that had been seeded the day before and cultured for 6 h at a 37°C incubator with 5% CO2. After incubation, Huh7 cells were washed with PBS and lysed in 0.1% TritonX-100 in PBS, and then incubated on a shaker for 10 min at room temperature. The luciferase activity of the cell lysates was measured as detailed above.
To determine the distribution of EVs containing Cas9-Nluc-CRY2 in mice after tail vein injection, tissues were harvested and lysed in 0.1% TritonX-100 using the Qiagen Tissue Lyser II according to the manufacturer’s instructions. The tissue lysates were diluted to a suitable concentration and mixed with buffer-diluted Nano-Glo substrate (Promega). The luciferase activity of the tissue lysates was measured as detailed above [24 (link)].
To detect the luciferase activity of EVs uptake by recipient cells, isolated EVs were added to Huh7 cells that had been seeded the day before and cultured for 6 h at a 37°C incubator with 5% CO2. After incubation, Huh7 cells were washed with PBS and lysed in 0.1% TritonX-100 in PBS, and then incubated on a shaker for 10 min at room temperature. The luciferase activity of the cell lysates was measured as detailed above.
To determine the distribution of EVs containing Cas9-Nluc-CRY2 in mice after tail vein injection, tissues were harvested and lysed in 0.1% TritonX-100 using the Qiagen Tissue Lyser II according to the manufacturer’s instructions. The tissue lysates were diluted to a suitable concentration and mixed with buffer-diluted Nano-Glo substrate (Promega). The luciferase activity of the tissue lysates was measured as detailed above [24 (link)].
