Summary of Study ST003607

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002231. The data can be accessed directly via it's Project DOI: 10.21228/M87R79 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003607
Study Title	Global metabolomics and tracing of 13C-labeled metabolites of bone marrow derived macrophages following engulfment of dead 13C-labeled Citrobacter rodentium, Listeria innocua, Staphilococcus aureus, or Pseudomonas aeruginosa.
Study Summary	Global metabolomics and 13C-tracing analysis of bone marrow derived macrophages exposed to heat-killed uniformly 13C-labelled Citrobacter rodentium, Listeria innocua, Staphilococcus aureus or Pseudomonas aeruginosa for 4h.
Institute	University of Colorado Anschutz Medical Campus
Last Name	Haines
First Name	Julie
Address	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email	julie.haines@cuanschutz.edu
Phone	3037243339
Submit Date	2024-11-26
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-12-27
Release Version	1

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Treatment:

Treatment ID:	TR003745
Treatment Summary:	Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were generated in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Gibco). BMDMs were used on day 5 to 7 after seeding. Period of differentiation of the cells, concentration of cells when replating and time-lapse between replating and stimulation with bacteria were important parameters to maintain metabolic backgrounds homogenous between experiments. Preparation of viable and killed U-[13C]Bacteria: Citrobacter rodentium, Staphylococus aureus, Listeria innocua, Pseudomonas aeruginosa were grown overnight with shaking in LB, diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts before addition to cells. For labeling, 10 µl of an overnight cultured of bacteria was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, and 0.5% U-13C glucose (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For antibiotic killing, bacteria were incubated for 6h to 12h with streptomycin or gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. Treatment of macrophages: 2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated 6-well plate (BD Falcons). Cells were then stimulated with killed labelled bacteria at MOI 25, centrifuged at 2000 rpm for 5 min. BMDMs were incubated for 5 min and washed with PBS to remove non-ingested bacteria and further incubated for 4h.

Treatment ID:

TR003745

Treatment Summary:

Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were generated in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Gibco). BMDMs were used on day 5 to 7 after seeding. Period of differentiation of the cells, concentration of cells when replating and time-lapse between replating and stimulation with bacteria were important parameters to maintain metabolic backgrounds homogenous between experiments. Preparation of viable and killed U-[13C]Bacteria: Citrobacter rodentium, Staphylococus aureus, Listeria innocua, Pseudomonas aeruginosa were grown overnight with shaking in LB, diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts before addition to cells. For labeling, 10 µl of an overnight cultured of bacteria was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, and 0.5% U-13C glucose (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For antibiotic killing, bacteria were incubated for 6h to 12h with streptomycin or gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. Treatment of macrophages: 2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated 6-well plate (BD Falcons). Cells were then stimulated with killed labelled bacteria at MOI 25, centrifuged at 2000 rpm for 5 min. BMDMs were incubated for 5 min and washed with PBS to remove non-ingested bacteria and further incubated for 4h.