Metabolomics Workbench : NIH Data Repository

Return to study ST002047 main page

MB Sample ID: SA192464

Local Sample ID:	OPCO_NN_20210318220941
Subject ID:	SU002129
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002129
Subject Type:	Cultured cells
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
OPCO_NN_20210318220941	SA192464	FL023652	OPC Over	Factor

Collection:

Collection ID:	CO002122
Collection Summary:	RGC cell isolation. Primary retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning protocol described in a previous study (Dvoriantchikova, Degterev et al., 2014). Briefly, whole retinas of 10 postnatal day 11-12 pups were incubated in papain solution (16.5 U/mL; Worthington Biochemical, LS003127) for 30 minutes. Macrophaged and endothelial cells were removed from the cell suspension by panning with anti-macrophage antiserum (Accurate Chemical, AIA31240). RGCs were bound to the panning plates containing the antibody against Thy1.2 (VWR, 102646-060) and were then released by incubation with trypsin solution (Sigma, T9935). RGCs were plated in a plate coated with poly-d-lysine (Sigma, P6407) and Laminin (Sigma, L-6274). RGCs were either not treated (addition of RGC growth media), treated with LPC 18:0 at 10 µM in RGC growth media, or treated with LPC 18:1 at 10 µM in RGC growth media. RGCs were incubated for 24 hours and harvested the next day. OPC cell culture. Oligodendrocyte Precursor Cells (OPC) were purchased from ScienceCell Research Laboratories (R1600, Carlsbad, CA 92008) and cultured according to manufacturer’s recommendations. In brief, plates were coated with poly-d-lysine (Sigma, P6407; 2 µg/cm2) and incubated overnight at 37ºC. OPC growth media was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC growth supplement (ScienceCell Research Laboratories, 1652) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). OPC differentiation medium was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC differentiation supplement (ScienceCell Research Laboratories, 1672), fetal bovine serum (ScienceCell Research Laboratories, 0005) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). Poly-d-lysine coated plate was washed twice with sterile water, OPC growth media was added to each well and plate was placed in incubator to equilibrate for 15 minutes (37ºC and 5% CO2). Frozen vial containing OPCs was warmed in 37ºC water bath and gently rotated to equally suspend cells. 15,000 cells/cm2 were seeded and left overnight (~16 to ~18 hours) in incubator. Media was changed daily for 2 days. OPCs were either not treated (addition of OPC growth media), differentiated (addition of OPC differentiation media), treated with LPC 18:0 at 10 µM in OPC growth media, or treated with LPC 18:1 at 10 µM in OPC growth media. OPCs were incubated for 24 hours and harvested the next day. OPCs and oligodendrocytes were further validated using mRNA analysis following published methods (Jäkel, Agirre et al., 2019).
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR002141
Treatment Summary:	siRNA and overexpression. Cell transfection was carried using the INTERFERin transfection kit following manufacture’s recommendations (Polyplus Transfection, 89129-930). siRNA for LPCAT1, PLA2G4C, and LIPC were purchased from Ambion (Ambion, s102346, s107315, s67780 respectively), LPGAT1 and LIPC overexpression constructs were purchased from Genecopoeia (Genecopoeia, EX-mm15104-M61, EX-mm03119-M61). In brief, cells were transfected with 2 nM of siRNA or DNA in serum free cell culture media. INTERFERin reagent was added to the mix incubated at room temperature for 10 minutes and added to newly changed cell culture media for each well. Cells were incubated for 48 to 96 hours before harvesting.

Treatment ID:

TR002141

Treatment Summary:

siRNA and overexpression. Cell transfection was carried using the INTERFERin transfection kit following manufacture’s recommendations (Polyplus Transfection, 89129-930). siRNA for LPCAT1, PLA2G4C, and LIPC were purchased from Ambion (Ambion, s102346, s107315, s67780 respectively), LPGAT1 and LIPC overexpression constructs were purchased from Genecopoeia (Genecopoeia, EX-mm15104-M61, EX-mm03119-M61). In brief, cells were transfected with 2 nM of siRNA or DNA in serum free cell culture media. INTERFERin reagent was added to the mix incubated at room temperature for 10 minutes and added to newly changed cell culture media for each well. Cells were incubated for 48 to 96 hours before harvesting.

Sample Preparation:

Sampleprep ID:	SP002135
Sampleprep Summary:	15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile].

Sampleprep ID:

SP002135

Sampleprep Summary:

15 µg of total protein from homogenate sample were added to 4 times the volume of acetone at 20°C and incubated at 20°C overnight. Samples were centrifuged at 21,000 g at 4°C for 30 minutes (Thermo Fisher Megafuge 8R). Supernatant was discarded and pellet (very small) was air dried for 10 minutes. The pellet was re-suspended, denatured and reduced with 6 M urea, 10 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature. Following denaturing and reduction, the re-suspended pellet was alkylated with 15 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 minutes while maintained in darkness. Alkylation reaction was quench using 20 mM dithiothreitol in 50 mM ammonium bicarbonate for 1 hour at room temperature while kept in darkness. Sample was diluted using 50 mM ammonium bicarbonate to contain 1 M urea. Sample was digested using either trypsin or chymotrypsin (Promega, V5111, V106A) at a 1:30 (w/w) ratio of enzyme to protein. Digestion was incubated overnight at 37°C. Reaction was terminated using 50% formic acid at a 5:100 (v/v) ratio of formic acid to sample volume. Samples were stored at -20°C or immediately desalted. Samples were desalted using the Pierce Graphite Spin Columns (Thermo, 88302) following manufacturer’s recommendations. Samples were then evaporated using the CentriVap Concentrator system (Labconco, Kansas City, MO 64132-2696) and resuspended in 30 µl of protein resuspension solution [2% (v/v) acetonitrile, 0.1% (v/v) formic acid in mass spectrometry grade water]. Lipids were harvested by adding 400 µl of 1:1 v/v methanol/chloroform mix to 200 µl of cell culture lysate. Lysate was vortexed and incubated in ice for 5 minutes, followed by the addition of 350 µl of chloroform. Lysate was centrifuged at 18,000g for 15 minutes at 4ºC. The organic layer containing lipids (bottom layer) was transferred to a new tube and desiccated using the CentriVap Concentrator system. Lipids were resuspended in 50 µl of lipid resuspension solution [50% v/v isopropyl alcohol and 50% v/v acetonitrile].

Combined analysis:

Analysis ID	AN003334
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Accela 600
Column	Thermo Acclaim C30 (150 x 2.1mm,3um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	μg/ml

Chromatography:

Chromatography ID:	CH002469
Instrument Name:	Thermo Accela 600
Column Name:	Thermo Acclaim C30 (150 x 2.1mm,3um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003104
Analysis ID:	AN003334
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Lipids were identified using LipidSearch software v. 4.1.
Ion Mode:	UNSPECIFIED