was used on the selected monoclonal cell population after lentiviral particle transfection.
We obtained more than 95% lentiviral transduction efficiency of AD-MSCs according to the selection of puromycin
as a beneficial agent for ideal therapeutic purposes .
Twenty-four hours later, puromycin
(2 [micro]g/mL) was added.
After 48 h incubation, cells were replenished every 2-3 days with fresh complete DMEM/F12 medium containing 1 [micro]g/ml puromycin
. After a week of puromycin
selection, cells in each well were expanded to a 10 cm cell culture plate for another week.
Linearized targeting construct (10 [micro]g, TakaRa, Shiga, Japan) and Each TALEN expression plasmid (15[micro]g) were electroporated to passage one cells (5x[10.sup.6]), and then seeded in 6-well plates (1.5x 105 cells/well) and puromycin
selection (1 [micro]g/mL) was applied at 24h post-seeding.
The resulting virus-containing supernatant was then used to infect EML cells, and the positive cells were screened using the dual markers for GFP and puromycin
After 48 h incubation, antibiotic selection medium ([alpha]-MEM growth medium with 10 [micro]g/ml puromycin
) was used to kill all the untransduced cells.
To eliminate nontransduced cells, puromycin
(Sigma-Aldrich, USA) was added to the medium at a final concentration of 4 [micro]g/mL , and the medium was incubated for at least 72 h.
A growing number of studies have also shown that MtD plays an essential role in rodent FSGS models for puromycin
aminonucleoside nephrosis (PAN) and aldosterone-induced renal injury [4,5].
GD25 [[alpha].sub.2][[beta].sub.1] integrin cells were kept under selection pressure with the addition of 10 [micro]g/mL puromycin
After 48 h, transfectants were diluted in 1:10 ratio, and the antibiotic selection was performed in a medium containing 5 [micro]g/ml puromycin
(Life Technologies, USA) for 4 weeks.