Table III.
Reporter Luciferase (Luc) DNA Loading Efficiency and Total Protein Content in Luc-EXOs and Luc-MVs Derived from hCMEC/D3 Cells
| Type of carrier | Amount of DNA transfected into cells (μg)a |
Amount of DNA in isolated EVs (ng) |
DNA loading (%)b | Total EV protein content (μg)c |
|---|---|---|---|---|
| EXOs | 0 | 381.6 ± 42.0 | N/A | 181.7 ± 70.5 |
| 12 | 480.8 ± 69.9 | 0.83 ± 0.23 | 172.0 ± 62.4 | |
| 24 | 390.8 ± 46.9 | 0.04 ± 0.02 | 187.0 ± 61.8 | |
| MVs | 0 | 33.6 ± 1.8 | N/A | 155.7 ± 63.0 |
| 12 | 60.0 ± 7.4 | 0.22 ± 0.08 | 152.3 ± 48.0 | |
| 24 | 81.5 ± 5.5 | 0.20 ± 0.02 | 192.4 ± 69.1 |
The amount of DNA transfected into cells indicates the total amount of Luc pDNA that was transfected into the donor cells using Lipofectamine 3000; EVs were isolated from a single 24-well plate that was transfected with a total of either 0, 12, or 24 μg pDNA.
The %DNA loading values were calculated after subtracting the fluorescence values obtained from the 0 μg/well DNA-transfected cells (naïve EVs) from the pDNA-loaded EVs (pDNA-EVs). The obtained values were divided by the amount of DNA transfected into cells and multiplied by 100 (Eq. 1).
The total protein amount in pDNA-EXOs or MVs was measured using a MicroBCA assay. Data are presented as mean ± SE of four independent experiments (n = 3/experiment)