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5 protocols using u 13c acetate

1

Tracing Metabolic Flux in Glucose and Acetate

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To monitor carbon flux during growth in glucose, cells were
cultured in minimal medium with 0.3% unlabeled glucose to
OD600=0.4 in an anaerobic chamber heated to
37°C and transferred to a prewarmed filter membrane as described
for metabolomics extractions. Medium switch was performed under
continual flow of minimal medium with 0.3% of 100%
[U-13C]-glucose (Cambridge Isotope
Laboratories CLM-1396-PK) for up to 5 minutes, and metabolites were
extracted after 10, 20, 40, 80, 160 and 320 seconds as described above
(without the spike-in of the 13C labeled internal standard).
The acetate growth shift experiment was conducted following the same
procedure, with the cells grown in minimal medium containing
0.3% unlabeled glucose and 0.1% unlabeled acetate and
switched to medium containing 0.3% unlabeled glucose and
0.1% of 100% [U-13C]-acetate
(Cambridge Isotope Laboratories).
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2

Metabolic Labeling Techniques in Cell Culture

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Collagen type 1 (rat tail) was from Millipore or from PureCol® (bovine) (Advanced Biomatrix, USA). The CPT1a inhibitor (+)-etomoxir sodium salt hydrate was purchased from CNIO Carlos III Therapies. Mitomycin C (MitoC), sodium palmitate, dimethyl sulfoxide (DMSO), NAC, sodium acetate, oligomycin, cycloheximide, cytidine, adenine, guanosine, methotrexate, carnitine, dNTP mix and tamoxifen were from Sigma-Aldrich (Bornem, Belgium). 5-fluorouracil (TEVA Pharma Belgium) was obtained from the pharmacy of the university hospital Leuven. Nucleoside mix was from Millipore (Belgium)15 . Hoechst 33342 and L-homopropargylglycine (HPG) were from Molecular Probes and L-glutamine and penicillin/streptomycin were from Gibco® (Invitrogen, Life Technologies, Ghent, Belgium). Uniformly labeled [U-13C]-potassium palmitate, [U-13C]-acetate, [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-algal fatty acid mix were obtained from Cambridge isotope laboratories, Inc. [U-14C]-palmitate, [9,10-3H]-palmitate, [6-14C]-D-glucose, [8-14C]-hypoxanthine and [3H]-thymidine were from Perkin Elmer.
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3

Metabolic Labeling Techniques in Cell Culture

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Collagen type 1 (rat tail) was from Millipore or from PureCol® (bovine) (Advanced Biomatrix, USA). The CPT1a inhibitor (+)-etomoxir sodium salt hydrate was purchased from CNIO Carlos III Therapies. Mitomycin C (MitoC), sodium palmitate, dimethyl sulfoxide (DMSO), NAC, sodium acetate, oligomycin, cycloheximide, cytidine, adenine, guanosine, methotrexate, carnitine, dNTP mix and tamoxifen were from Sigma-Aldrich (Bornem, Belgium). 5-fluorouracil (TEVA Pharma Belgium) was obtained from the pharmacy of the university hospital Leuven. Nucleoside mix was from Millipore (Belgium)15 . Hoechst 33342 and L-homopropargylglycine (HPG) were from Molecular Probes and L-glutamine and penicillin/streptomycin were from Gibco® (Invitrogen, Life Technologies, Ghent, Belgium). Uniformly labeled [U-13C]-potassium palmitate, [U-13C]-acetate, [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-algal fatty acid mix were obtained from Cambridge isotope laboratories, Inc. [U-14C]-palmitate, [9,10-3H]-palmitate, [6-14C]-D-glucose, [8-14C]-hypoxanthine and [3H]-thymidine were from Perkin Elmer.
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4

Isotopic Tracing of Parasite Metabolism

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RH and ΔACL/iΔACS intracellular parasites were incubated in the absence of Shld-1 for 16 h and simultaneously labelled with either 10 mM U-13C-glucose or 2 mM U-13C-acetate (Cambridge Isotope Laboratories). Per sample, 108 parasites were isolated and purified as described above. FAs were extracted, derivatised and analysed as described previously [16 (link)]. Fatty acid methyl esters (FAMEs) were identified based on retention times and the library integrated in Xcalibur (Thermo Fisher Scientific). The abundance was determined based on the peak intensity relative to the cholesterol signal intensity in each sample. Abundance of the C14:0 and C26:1-FAME mass isotopologues (m/z 242–256; m/z 376–390) was determined using Xcalibur (Thermo Fisher Scientific) and OpenChrom software. The extent of 13C-labelling was determined using the Excel (Microsoft) software following correction for the occurrence of natural isotopes as described by Zamboni et al. [83 (link)]. Abundance data represent the average of 6 biological replicates, and labelling data represents the average of 3 biological replicates. Statistical significance of differences in labelling and abundance were assessed by t-test. Standard deviation and p values are indicated in the figure.
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5

Isotope-labeled Metabolite Extraction

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LC–MS grade water (H2O) was purchased from Honeywell (Muskegon, MI, USA). LC–MS grade acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). Ammonium hydroxide (NH4OH) and ammonium acetate (NH4OAc) were purchased from Sigma (St. Louis, MO, USA). The [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-acetate was purchased from Cambridge Isotopes laboratories (MA, USA).
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