Alkaline phosphatase blue membrane substrate solution

For AP staining, 5000 cells (wt and Mettl6 KO clones #D1 and #G6) were seeded, and after 5 days, cells were fixed (4% formaldehyde) and stained for AP with Alkaline Phosphatase Blue Membrane Substrate Solution (Sigma-Aldrich, AB0300), according to the manufacturer’s protocol. mESCs were cultured on 0.2% gelatin-coated dishes in DMEM (Sigma-Aldrich) supplemented with 16% FBS (Sigma-Aldrich), 0.1 mM β-mercaptoethanol (Invitrogen), 2 mM l-glutamine (Sigma-Aldrich), 1× MEM nonessential amino acids (Sigma-Aldrich), penicillin (100 U/ml), streptomycin (100 μg/ml; Sigma-Aldrich), and homemade recombinant LIF tested for efficient self-renewal maintenance.

For reprogramming to iPSCs, we used three different systems: i4F MEFs were cultured in the presence of doxycycline at a concentration of 1 μg/mL, and medium was replaced every 48 h during 14 days; WT MEFs were transduced with lentiviral plasmids Tet-O-FUW-OSKM and FUW-M2rtTA and cultured with doxycycline as above; or WT MEFs were transduced with retroviral plasmids: pMXs-Oct4, pMXs-Sox2, pMXs-Klf4, and pMXs-c-Myc (see Supplemental Experimental Procedures for plasmid details), and cultured in iPSC medium.
After 14 days, cell cultures were fixed with 4% paraformaldehyde and the AP activity was detected with Vector Red Substrate Kit (Vector Labs) or Alkaline Phosphatase Blue Membrane Substrate Solution (Sigma-Aldrich) according to the manufacturer's instructions. Plates were scanned with Epson Perfection V550 Photo (Epson) and quantification of positive colonies was performed using ImageJ software.