Nbt bcip substrate solution

ALP activity was measured using staining and spectrophotometry. At day 7 of culturing, the cells were washed with PBS and then fixed with 10% formalin for 5 min at room temperature. Then, the cells were rinsed with PBS three times and stained with NBT/BCIP substrate solution (Roche Diagnostics, Mannheim, Germany) in incubation for 10 min at 37 °C. The cells were washed with deionized water after aspirating the staining solution.
To measure ALP activity, at day 7 of culturing the cells were washed and lysed with sodium carbonate buffer (25 mM, pH 10) supplemented with 1.0% triton X-100. ALP activity in the supernatant of the lysate was measured according to our previous method [25 (link)].

∼400,000 MEFs bearing an OKSM polycistronic cassette under the control of a Tet-responsive element (TetO) inserted in the 3΄-UTR of Col1a1 locus (originating from the transgenic murine strain B6;129S4-Col1a1^tm1(tetOCol1a1^{Tm1TetO-Pou5f1,-Klf4,-Sox2,-Myc)Hoch}/J, Jackson ID, Cat. No. 011001) [52 (link)] were seeded in 10 cm plates. For the induction of OSKM expression, doxycycline was added at a 2 μg/ml final concentration. At day 7 of the reprogramming process, cells were growing in MEF media and then were switched to ESC media.
For reprogramming efficiency assays, transduced cells at the 6^th day of reprogramming were trypsinized to single-cell suspensions and ∼75,000 cells from each culture were seeded onto gelatin 0.1% pre-coated cell culture dishes, with Mitomycin C-treated MEFs being used as feeder layers. The cultures were maintained in mESC medium. On days 18 to 21, all cultures were stained for Alkaline Phosphatase (AP) activity, using the NBT/BCIP substrate solution (Roche Life Sciences, Cat. No. 11681451001), in NTMT buffer [100mM Tris-HCl, 100mM NaCl, 50 mM MgCl₂ and 0.1% Tween-20, pH 9.5] and counted. Reprogramming Efficiency (RE) (%) was calculated by dividing the total number of AP-stained formations with the number of trypsinized cells seeded on day 6 on the feeder layers (∼75,000 cells) and multiplying by 100.

Plasmid DNA was digested with 50 U of Hind III (New England Biolabs) for 1 h at 37°C and separated by electrophoresis for 3 h at 45 V in 0.8% agarose. After migration, the digested plasmids were transferred to positively charged nylon membranes (Roche Diagnostics, Mannheim, Germany) using a vacuum blotter model 785 (Bio-Rad). The membranes were probed with digoxigenin labelled PCR products for the genes aad(D), lnu(A), spc, tet(K) and tet(M) [46 (link)] using the PCR DIG probe synthesis kit (Roche Diagnostics) (S1 Table). Pre-hybridizations and hybridizations were carried out at 65°C for 30 min and 18 h, respectively, in hybridization buffer with subsequent washes, as recommended by the manufacturer. To detect the presence of digoxigenin-labelled probes, the colorimetric method (NBT/BCIP substrate solution, Roche Applied Science) was used. PCR products were used as hybridization control and control DNA DIG-labelled as detection control.