X non essential amino acids

A3 and B2 ESC were plated at 10,000 cells/cm² on 50 ng/mL of collagen IV-coated or fibronectin-coated plates (BD Biosciences) and cultured in Stage 1 Differentiation Medium, called NSD12b, for A3 and B2, respectively.24 (link) Briefly, our serum-free Stage 1 Differentiation Medium, preoptimized for B2 and A3 cells included alpha-DMEM (Invitrogen), 15% KSR (Invitrogen), 1x penicillin-streptomycin (Invitrogen), 1x nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (Calbiochem), 5 ng/mL of bone morphogenetic protein-4 (R&D Systems) and 0 or 20 ng/mL of VEGF (R&D Systems) for A3 and B2, respectively. At 0, 1, 2, 3, and 4 days, the cells were collected and stained for upregulation of mesoderm marker, Flk-1, and downregulation of pluripotent stem cell marker, Oct3/4. Briefly, cells were fixed 4% paraformaldehyde overnight before being placed in a solution of 1% bovine serum albumin (BSA) (Sigma), 0.7% Triton X-100 (MP Biomedicals LLC), and 5% donkey serum (Fitzgerald) for 1 hour prior to staining. Cells were stained with Alexa Fluor 647 anti-mouse Flk-1 (Biolegend), or anti-Oct3/4 PE conjugated antibody (Becton Dickinson [BD]) overnight. Cells were rinsed and analyzed on an LSR II flow cytometer (BD). All results were gated to 5% of an isotype control.