Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA170658

Local Sample ID:	M2d_038_081418
Subject ID:	SU001912
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

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Subject:

Subject ID:	SU001912
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
M2d_038_081418	SA170658	FL020537	24	hours
M2d_038_081418	SA170658	FL020537	IL6_LIF	treatment

Collection:

Collection ID:	CO001905
Collection Summary:	CD14+ PBMCs were isolated from whole human blood, differentiated into macrophages with M-CSF, followed by polarization stimuli for 24-72hrs. Cells were counted, washed with PBS, and immediately frozen in liquid nitrogen.
Sample Type:	Blood (whole)

Treatment:

Treatment ID:	TR001925
Treatment Summary:	M0 macrophages were treated only with M-CSF; M1 macrophages were treated with LPS & IFN-gamma; M2a macrophages were treated with IL-4 & IL-13; M2b macrophages were treated with LPS & ICs; M2c macrophages were treated with IL-10; M2d macrophages were treated with IL-6 & LIF. Treatments course was 24hr or 72hr.

Sample Preparation:

Sampleprep ID:	SP001918
Sampleprep Summary:	With the goal of creating a comprehensive snapshot of cellular function, multiple experimental sample types were collected from each MΦ phenotype during cell harvest to obtain transcriptomic, proteomic, and metabolomic data from each experimental trial. At the desired time point, the cellular supernatant, composed of cell culture media and any non-adherent cells, was removed from the cell culture well and placed into falcon tubes on ice. One mL of ice-cold phosphate buffered saline (PBS) was added to the adherent cell layers and then removed to the tubes containing the cellular supernatant and the tubes centrifuged at 2000 rpm for 8 minutes. The collected supernatant was stored at -80° C until further analysis. Pelleted cells were flash-frozen in liquid nitrogen and stored on ice, while the remaining adherent cells were quenched with 350 µL of ice-cold 100% methanol (Honeywell; Muskegon, MI, USA) and 350 µL of ice-cold ultrapure distilled water (Invitrogen; Grand Island NY, USA) and removed through gentle cell scraping and added to the cell pellet fraction. Following the addition of 700 µL of ice-cold chloroform (Acros Organics; Thermo Fisher Scientific; Waltham, MA, USA), the collected cells were vortexed for 30 seconds and were transferred to FastPrep® lysing matrix D tubes (MP Biomedicals; Auckland, New Zealand). To achieve cell lysis, the tubes were homogenized during two cycles of 40 s each at 4.0 m/s with a 90 s delay between cycles utilizing the FastPrep-24™ 5G Homogenizer (MP Biomedicals; Auckland, New Zealand). The homogenized samples were centrifuged at 16,000 x g for 5 minutes at 4° C, and then placed immediately on ice. The polar (methanol/water) layer and non-polar (chloroform) layers were subsequently transferred to 1.5 mL protein low binding microcentrifuge tubes. These metabolite suspensions were lyophilized overnight without heat on a Thermo Scientific™ Savant™ ISS110 SpeedVac™ (Waltham, MA, USA) and stored at -80°C until the samples were shipped to Metabolon for further analysis. The remaining interphase layer was flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Sampleprep ID:

SP001918

Sampleprep Summary:

With the goal of creating a comprehensive snapshot of cellular function, multiple experimental sample types were collected from each MΦ phenotype during cell harvest to obtain transcriptomic, proteomic, and metabolomic data from each experimental trial. At the desired time point, the cellular supernatant, composed of cell culture media and any non-adherent cells, was removed from the cell culture well and placed into falcon tubes on ice. One mL of ice-cold phosphate buffered saline (PBS) was added to the adherent cell layers and then removed to the tubes containing the cellular supernatant and the tubes centrifuged at 2000 rpm for 8 minutes. The collected supernatant was stored at -80° C until further analysis. Pelleted cells were flash-frozen in liquid nitrogen and stored on ice, while the remaining adherent cells were quenched with 350 µL of ice-cold 100% methanol (Honeywell; Muskegon, MI, USA) and 350 µL of ice-cold ultrapure distilled water (Invitrogen; Grand Island NY, USA) and removed through gentle cell scraping and added to the cell pellet fraction. Following the addition of 700 µL of ice-cold chloroform (Acros Organics; Thermo Fisher Scientific; Waltham, MA, USA), the collected cells were vortexed for 30 seconds and were transferred to FastPrep® lysing matrix D tubes (MP Biomedicals; Auckland, New Zealand). To achieve cell lysis, the tubes were homogenized during two cycles of 40 s each at 4.0 m/s with a 90 s delay between cycles utilizing the FastPrep-24™ 5G Homogenizer (MP Biomedicals; Auckland, New Zealand). The homogenized samples were centrifuged at 16,000 x g for 5 minutes at 4° C, and then placed immediately on ice. The polar (methanol/water) layer and non-polar (chloroform) layers were subsequently transferred to 1.5 mL protein low binding microcentrifuge tubes. These metabolite suspensions were lyophilized overnight without heat on a Thermo Scientific™ Savant™ ISS110 SpeedVac™ (Waltham, MA, USA) and stored at -80°C until the samples were shipped to Metabolon for further analysis. The remaining interphase layer was flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Combined analysis:

Analysis ID	AN002977
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	raw area count

Chromatography:

Chromatography ID:	CH002206
Chromatography Summary:	Low pH polar
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002767
Analysis ID:	AN002977
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Proprietary analytical software for integration and peak picking
Ion Mode:	POSITIVE