Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA159040

Local Sample ID:	IR_15_60120
Subject ID:	SU001785
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU001785
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
IR_15_60120	SA159040	FL018518	SENCells_IR	Factor

Collection:

Collection ID:	CO001778
Collection Summary:	Cell culture: Human fetal lung fibroblasts (IMR-90) and HEPG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM - Gibco) supplemented with 10% FBS and penicillin/streptomycin (Gibco). HUVEC cells were obtained from the ATCC and cultured using the ATCC protocol and culture media. Quiescence was induced by replacing culture media with media containing 0.2% FBS for 72 h before analysis. All cells were cultured at 3% O2, and used prior to 40 population doublings, other than replicative senescent cells, which were cultured until replicative exhaustion. All cells were mycoplasma free. Mouse Plasma: Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute. For DOXO treatments, 10-16 wk old p16-3MR mice received one intraperitoneal (i.p.) injection of 10 mg/kg of doxorubicin hydrochloride in PBS, and treated 5 d later with GCV or vehicle. GCV was administered via daily i.p. injections for 5 consecutive days at 25 mg/kg in PBS. Control mice were injected with an equal volume of PBS. Mice were euthanized and tissues collected 10 d after DOXO challenge. For aging studies, C57BL/6 mice were aged for 6 or 24 mo, at which point mice were euthanized and tissues collected for analysis. For the biomarker studies, mice with challenged with DOXO as above, and given either vehicle or ABT-263 6 weeks after DOXO challenge. Whole blood was collected by cardiac puncture into EDTA-tubes (lavender caps – BD Biosciences) and spun at 2000g for 5 minutes. For urine collection, mice were placed in plastic containers prior to euthanasia.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR001798
Treatment Summary:	Cell culture: Human fetal lung fibroblasts (IMR-90) and HEPG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM - Gibco) supplemented with 10% FBS and penicillin/streptomycin (Gibco). HUVEC cells were obtained from the ATCC and cultured using the ATCC protocol and culture media. Quiescence was induced by replacing culture media with media containing 0.2% FBS for 72 h before analysis. All cells were cultured at 3% O2, and used prior to 40 population doublings, other than replicative senescent cells, which were cultured until replicative exhaustion. All cells were mycoplasma free. Mouse Plasma: Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute. For DOXO treatments, 10-16 wk old p16-3MR mice received one intraperitoneal (i.p.) injection of 10 mg/kg of doxorubicin hydrochloride in PBS, and treated 5 d later with GCV or vehicle. GCV was administered via daily i.p. injections for 5 consecutive days at 25 mg/kg in PBS. Control mice were injected with an equal volume of PBS. Mice were euthanized and tissues collected 10 d after DOXO challenge. For aging studies, C57BL/6 mice were aged for 6 or 24 mo, at which point mice were euthanized and tissues collected for analysis. For the biomarker studies, mice with challenged with DOXO as above, and given either vehicle or ABT-263 6 weeks after DOXO challenge. Whole blood was collected by cardiac puncture into EDTA-tubes (lavender caps – BD Biosciences) and spun at 2000g for 5 minutes. For urine collection, mice were placed in plastic containers prior to euthanasia.

Treatment ID:

TR001798

Treatment Summary:

Cell culture: Human fetal lung fibroblasts (IMR-90) and HEPG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM - Gibco) supplemented with 10% FBS and penicillin/streptomycin (Gibco). HUVEC cells were obtained from the ATCC and cultured using the ATCC protocol and culture media. Quiescence was induced by replacing culture media with media containing 0.2% FBS for 72 h before analysis. All cells were cultured at 3% O2, and used prior to 40 population doublings, other than replicative senescent cells, which were cultured until replicative exhaustion. All cells were mycoplasma free. Mouse Plasma: Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute. For DOXO treatments, 10-16 wk old p16-3MR mice received one intraperitoneal (i.p.) injection of 10 mg/kg of doxorubicin hydrochloride in PBS, and treated 5 d later with GCV or vehicle. GCV was administered via daily i.p. injections for 5 consecutive days at 25 mg/kg in PBS. Control mice were injected with an equal volume of PBS. Mice were euthanized and tissues collected 10 d after DOXO challenge. For aging studies, C57BL/6 mice were aged for 6 or 24 mo, at which point mice were euthanized and tissues collected for analysis. For the biomarker studies, mice with challenged with DOXO as above, and given either vehicle or ABT-263 6 weeks after DOXO challenge. Whole blood was collected by cardiac puncture into EDTA-tubes (lavender caps – BD Biosciences) and spun at 2000g for 5 minutes. For urine collection, mice were placed in plastic containers prior to euthanasia.

Sample Preparation:

Sampleprep ID:	SP001791
Sampleprep Summary:	Protocol for Lipid Extraction (10.24.2019) Lipid Biomarker for Senolysis Study 1. 80% MeOH was added to each sample 500µL to plasma samples 100µL to urine sample 2. Transfer the contents to 9 mL glass tube and add 1.0 mL of CHCl3 (containing 200 ng/mL FA 17:0 Saturated). Vortex mix for 5 minutes. 3. Place the glass tube into a 15mL polypropylene culture tube with Kimi wipes inserted at bottom as a cushion. Wrap the culture tube with paraffin and place in centrifuge. This will prevent glass tube being shattered in the centrifuge. 4. Centrifuge at 4°C for 10 minutes 3000rpm. 5. After spinning there will be two distinct layers, the top layer is the aqueous layer containing the aqueous metabolites (80% MeOH) and bottom layer which contains lipids. 6. Transfer the lower layer into 1.5ml vials. 7. Transfer from the 1.5mL tube the exact amount of each sample into the MS/HPLC glass vial and dry down using nitrogen gas. 8. Reconstitute the lipids with 80µL chloroform. 9. Transfer upper layer (metabolites) into labeled vials and store at -80°C, if possible. Samples stored in -80°C. Sample volume submitted -Plasma samples=50µL Urine samples= 10µL Sample volume in each vial- Plasma samples µL and Urine samples µL -submitted to mass spec All samples were split in half ½ given to CW for further testing other 1/2 in -80°C

Sampleprep ID:

SP001791

Sampleprep Summary:

Protocol for Lipid Extraction (10.24.2019) Lipid Biomarker for Senolysis Study 1. 80% MeOH was added to each sample 500µL to plasma samples 100µL to urine sample 2. Transfer the contents to 9 mL glass tube and add 1.0 mL of CHCl3 (containing 200 ng/mL FA 17:0 Saturated). Vortex mix for 5 minutes. 3. Place the glass tube into a 15mL polypropylene culture tube with Kimi wipes inserted at bottom as a cushion. Wrap the culture tube with paraffin and place in centrifuge. This will prevent glass tube being shattered in the centrifuge. 4. Centrifuge at 4°C for 10 minutes 3000rpm. 5. After spinning there will be two distinct layers, the top layer is the aqueous layer containing the aqueous metabolites (80% MeOH) and bottom layer which contains lipids. 6. Transfer the lower layer into 1.5ml vials. 7. Transfer from the 1.5mL tube the exact amount of each sample into the MS/HPLC glass vial and dry down using nitrogen gas. 8. Reconstitute the lipids with 80µL chloroform. 9. Transfer upper layer (metabolites) into labeled vials and store at -80°C, if possible. Samples stored in -80°C. Sample volume submitted -Plasma samples=50µL Urine samples= 10µL Sample volume in each vial- Plasma samples µL and Urine samples µL -submitted to mass spec All samples were split in half ½ given to CW for further testing other 1/2 in -80°C

Combined analysis:

Analysis ID	AN002782
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 6520
Column	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type	ESI
MS instrument type	QToF
MS instrument name	Agilent 6520 QTOF
Ion Mode	NEGATIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH002058
Instrument Name:	Agilent 6520
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002578
Analysis ID:	AN002782
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QToF
MS Type:	ESI
MS Comments:	Full scan mode was used. Data processed using profinder software
Ion Mode:	NEGATIVE