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A 10 µL aliquot was taken from the reaction mixture and injected in HPLC (Agilent 1200 series, Waldbrunn, Germany) equipped with a C18 reverse-phase capillary column (250 × 4.6 mm, 5 µL) as well as an ultraviolet detector at wavelength range of 200 325 which was applied for DHCA evaluation.
Before imaging, animals were anesthetized in an acrylic chamber with 1.5% isofluorane/air mixture and injected i.p. with 40 mg/ml of luciferin potassium salt in PBS at a dose of 150 mg/kg body weight.
At this point, the mice were anesthetized (2% isoflurane/98% oxygen mixture) and injected intravenously with morpholino-[I]IPQA (2.6 MBq/mouse).
Before imaging, CB17/SCID mice were anesthetized in a chamber with a 1.5% (v/v) isoflurane/air mixture and injected i.p. with luciferin (150 mg/kg body weight).
The mice were anesthetized with a 1.5% isoflurane-oxygen mixture and injected intraperitoneally with luciferase potassium salt solution (Sigma-Aldrich) at a dose of 150 mg/kg body weight and imaged 10 min later.
After the reaction, a single aliquot of 50 μL was withdrawn from the gas phase on top of the reaction mixture and injected, using a gastight syringe, into a gas chromatograph for analysis.
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The water-immiscible extractant was collected by centrifugation, dried under low pressure, reconstituted in methanol water mixture (1 1), and injected into a HPLC system for the determination of CPAs.
Cells were suspended in a F12 (Invitrogen)/Matrigel (high concentration, BD Biosciences) mixture (4:1) and injected into the mammary fat pad in a 50 μl volume.
Pten:p53-mutant mouse (200,000 cells/injection) or human BT549 (1 million cells/injection) tumor cells were resuspended in 20 μl media/matrigel mixture (1 1) and injected into #4 mammary glands of NOD/SCID females (9 mice per group).
Briefly, single-cell suspensions were prepared in a PBS-Matrigel (BD Bioscience) mixture (v/v) and injected in a 100 μl volume subcutaneously in the back of CB17/SCID mice (Charles River Laboratories, L'Arbresle, France) anesthetized by i.p. injection of ketamine/xylasine (50 and 10 mg/kg, respectively).
A variety of sorted SP and non-SP cells (ranging from 102 to 106) were resuspended in 100 µL of a DMEM/Matrigel (Matrigel Basement Membrane Matrix; Becton Dickinson) mixture (1∶1 v/v) and injected subcutaneously into 6-week-old nude mice (Jackson Laboratory).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com