Please read Metabolite Processing Precautions
before proceeding.
⦁ Prepare and separate all material and solutions before you start. Check the Recommended Resources List.
⦁ Allow the cells thaw in ice.
⦁ Prepare two tubes labeled as QC (pooled quality control).
NOTE: One tube will be for the polar (QCp) fraction and the other for the non-polar fraction (QCnp). If only one LC method
is used, you may combine the fractions and in this case make only one QC tube.
⦁ Add 200 µL of ice-cold extraction solution A to the samples.
⦁ Homogenize the cells by beating for 10 s and cool down on ice for 30 s. Repeat the cycle 3 more times.
⦁ Centrifuge samples at 10,000 g for 10 min at 4°C.
⦁ Collect the supernatant into a new labeled tube (polar fraction).
⦁ Keep the tubes in dry ice and proceed to the next step.
⦁ IMMEDIATELY after collection of the polar fraction, add 1 mL of ice-cold extraction solution E (Non-polar extraction).
⦁ Repeat the homogenization step.
⦁ Centrifuge the mixture at 15000 g and 4°C for 10 min.
⦁ From the bi-phasic system created, transfer the upper layer to the tubes containing the polar fraction.
⦁ Carefully aspirate the bottom organic layer and transfer into another 2 mL Eppendorf tube.
⦁ From the polar fraction, take 10% of each sample and transfer to the QCp. Do the same procedure for non-polar fraction (QCnp tube).
⦁ Freeze-dry the polar fraction in a lypholyzer and the non-polar extract in the glass cupboard (lypholyzers ARE NOT compatible with chloroform).
⦁ PAUSE POINT Store the samples at -80 °C for up to 12 weeks.