Dissolve 11.51g in 800-900ml of ddH2O stirring gently until completely solubilized. Adjust pH of the medium to 0.1-0.3 pH unit below the desired level. To make complete HPLM, please add the Additional Polar Metabolites (listed below) not included in this powder to the listed concentrations. Then add additional water to bring the solution to 1L. Filter-sterilize using a 0.22 micron membrane filter. Aliquot into sterile containers. Do not autoclave. Contains heat-labile compounds that can be damaged with autoclaving. Component Concentration (uM) 2-hydroxybutyrate 50 3-hydroxybutyrate 50 Acetone 60 Acetylglycine 90 Alpha-aminobutyrate 20 Betaine 70 Carnitine 40 Citrulline 40 Creatine 40 Creatinine 75 Formate 50 Glycerol 120 Lactate 1600 Malonate 10 Phenol red 14

Store powdered media at RT. Stable for 12 months after receipt. Opened bottles should be capped tightly and kept in a dark, low humidity environment. Prepared media should be kept at 4°C and used within a short period of time.