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Vials were loaded into auto-sampler for chromatographic operation.
Upon thawing, the LoBind tube and one of the Nunc vials were loaded onto the assay plate with no treatment aside from dilution to MRD.
Vials were loaded sequentially into the reactor, which was programmed for fastest possible heating up to the chosen set temperature, either 180, 200 or 220°C.
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Briefly, 3 groups of 25 CSp or per flies were transferred into empty narrow vials, which were loaded into the RING apparatus.
One set of five vials each were loaded with two pieces of Whatman filter paper holding 400 μl of 5% (w/v) sucrose solution alone (controls), 400 μl of 5% sucrose solution with 10 mM paraquat or 400 μl of 5% sucrose solution with 15% (v/v) H2O2.
Samples were loaded in vials, flushed with helium, and dissolved in 99% phosphoric acid at 72°C for a minimum of one hour.
At the end of the incubation period the organic layer was transferred into eppendorf tubes, centrifuged to separate any residual medium left, and 200 μl were loaded on GC vials and analyzed by GC-MS for product identification and GC-FID for quantification.
The sample was reconstituted in 200 µl of mobile phase A and 100 µl were loaded onto two autosampler vials, one of which was archived at −80°C.
Excess solvent was evaporated off by heating at 70°C, to leave 10 μl of samples, which was transferred to glass vials ready to be loaded onto the nanoLC column.
Vials were capped, loaded into the Geno/Grinder and run at 1400 strokes/min for 20 s × 6, then incubated for 60 min at 65°C.
Vials were then loaded into the ThinPrep 2000 processor (Cytyc Corporation).
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