Forty-eight hours after infection, cells were treated with 8 [micro]g/ml puromycin
to select for a pool of puromycin-resistant clones.
Infected cells were quickly selected using puromycin
for 3-5 days before use.
Following 12 h of cell culture, puromycin
(0.5 [micro]g/mL) was added to the medium and stable clones were maintained in 1 [micro]g/mL puromycin
until distinct colonies appeared large enough for colony picking.
The human T-cell line, H9, was co-transfected with one plasmid containing the guide RNA sequences that has homology to the amino-terminus of the ccr5 gene and the CRISPR/Cas9 and a second plasmid containing a puromycin
To establish stable transfected cells (CaPan2-shEI, CaPan-2-shCTR, PANC-1-EI, and PANC-1-SCR), cells were grown in culture medium with 10 [micro]g/ml puromycin
GFP-positive transduced hAD-MSCs measured at 72 h after transduction (Figure 4(b)) and were selected for with 2.5 [micro]g of puromycin
Transfected cells were monitored daily and subject to media change and antibiotic selection with puromycin
(8 ug/mL) every 48 hours for 12-14 days.
The stable PPAR[alpha] silenced SW480 cells were selected by puromycin
. SW480 cells (1x[10.sup.6]) were injected subcutaneously in the nude mice.
The next day, selection of transduced cells was initiated with the addition of 2 [micro]g/ml puromycin
(InvivoGen, San Diego, CA, USA) into the culture medium.
The retrovirus-infected, IFN[alpha]-producing cells (IFN[alpha]-AF-MSCs) were selected due to their resistance to puromycin
. For further identification, IFN[alpha]-AF-MSCs were also subjected to stem cell surface marker test (CD90, CD105, CD73, HLA-ABC, CD34, CD14, CD45, and HLA-DR) by flow cytometry.
(2 [micro]g/ml) (Solarbio) was used to select for transfected cells.