M3148 25ml

Imaging buffer base [10% (m/v) glucose (G7021-100G, Sigma-Aldrich), 50 mM Tris-HCl, pH 8.0 (TR2016-050-80, Biosesang), and 10 mM NaCl], dilution buffer (50 mM NaCl and 10 mM Tris-HCl, pH 8.0) and GLOX [14 mg glucose oxidase (G2133-250KU, Sigma-Aldrich), 50 μl of catalase (C3155-50MG, Sigma-Aldrich), and 200 μl of dilution buffer] were prepared in advance. Then, right before starting experiment, imaging buffer was freshly prepared by 100 $\times$ diluting GLOX and 1% βME (M3148-25ML, Sigma-Aldrich) in imaging buffer base. Every sample was soaked in imaging buffer for inducing photoswitching of Alexa 647 dyes.

MEFs were prepared from E13.5 PHGDH^tetO/tetO, PHGDH^tetO/+ or PHGDH^+/+ embryos with the Rosa26-M2rtTA allele using standard protocols. MEFs were maintained in DMEM with pyruvate (VWR, 10–013-CV), 10% tet-free FBS (VWR Seradigm, Lot 120B14), 2 mM glutamine (Invitrogen, 25030–164), penicillin/streptomycin (VWR, 45000–652) and 3.5 μl beta-mercaptoethanol (Sigma-Aldrich, M3148-25ML) per 500 ml DMEM.