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MB Sample ID: SA169110

Local Sample ID:10
Subject ID:SU001897
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Not applicable

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Subject:

Subject ID:SU001897
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Not applicable

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
10SA169110FL020177APOE3/3 ACMCondition

Collection:

Collection ID:CO001890
Collection Summary:Astrocyte conditioned media (ACM) was obtained from immortalized astrocyte cell lines (a gift from Dr. David Holtzman) that were originally generated from primary astrocytes from P1-2 pups of APOE targeted replacement mice (42). The immortalized astrocytes were conditioned with Neurobasal media supplemented with B27, Glutamax-I, Normocin and 1% penicillin/streptomycin for 24 hours. This ACM was then collected, stored at -80ºC, and thawed prior to use. WT primary cortical neuronal cultures were obtained from embryonic day 17 (E17) C57Bl/6 embryos. Briefly, pregnant mice were euthanized by cervical dislocation and the embryo brains extracted. The meninges were carefully stripped off and the cortices dissected out. The cortices were then enzymatically dissociated in 0.25% Trypsin-EDTA and resuspended in Neurobasal media supplemented with B27, Glutamax-I, Normocin and 1% penicillin/streptomycin. Dissociated cells were counted and 300K neurons per well were plated directly into ACM in poly-D-lysine (PDL)-coated 6-well plates. ACM-treated neurons were then incubated at 37⁰C for 7 days in a humidified chamber with 5% CO2, with 50% media exchange (with newly thawed ACM) once every 3 days.
Sample Type:Neurons

Treatment:

Treatment ID:TR001910
Treatment Summary:WT neurons were incubated for 7 days in either no ACM, APOE3/3 ACM, or APOE4/4 ACM.

Sample Preparation:

Sampleprep ID:SP001903
Sampleprep Summary:Lipid and small-molecule metabolite extraction was performed using a methyl tert-butyl ether (MTBE)/methanol extraction protocol. Briefly, harvested neurons were homogenized in ice-cold methanol. Following homogenization, samples were incubated in 1200 ul of MTBE for 1 hr at room temperature to separate organic-soluble lipids from aqueous-soluble lipids and other small-molecules. Finally, 360 ul of ultrapure water was added (for a final ratio of 3:1:0.9 MTBE:methanol:water) to resolve the two liquid phases, and each samples were centrifuged at 10,000 x g for 10 min. For this experiment, the upper organic phase was collected from each sample and stored in a separate tube, and the remaining protein pellets were resuspended in 25 mM ammonium bicarbonate, pH 8, with 2.5% SDS. A BCA protein assay was performed on each protein fraction, and the organic phase was normalized to their protein concentration equivalent with 100% methanol. All samples were then stored at -80°C prior to analysis.

Combined analysis:

Analysis ID AN002954 AN002955 AN002956
Analysis type MS MS MS
Chromatography type Reversed phase Reversed phase HILIC
Chromatography system Agilent 1260 Agilent 1260 Agilent 1260
Column Agilent Eclipse XDB-C18 (100 x 3.0mm) Agilent Eclipse XDB-C18 (100 x 3.0mm) Phenomenex Luna NH2 (150 x 2.1mm,3um)
MS Type ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name Agilent 6490 QQQ Agilent 6490 QQQ Agilent 6490 QQQ
Ion Mode NEGATIVE POSITIVE POSITIVE
Units area under the curve area under the curve area under the curve

Chromatography:

Chromatography ID:CH002188
Chromatography Summary:Reverse phase
Instrument Name:Agilent 1260
Column Name:Agilent Eclipse XDB-C18 (100 x 3.0mm)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002189
Chromatography Summary:Normal phase
Instrument Name:Agilent 1260
Column Name:Phenomenex Luna NH2 (150 x 2.1mm,3um)
Chromatography Type:HILIC

MS:

MS ID:MS002744
Analysis ID:AN002954
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Lipid profiling was performed using an Agilent 1260 HPLC coupled to an Agilent 6490 triple quadrupole (QQQ) mass spectrometer (41). Each sample was run through three separate chromatographic conditions (reverse-phase negative mode, reverse-phase positive mode and normal-phase positive mode) for the effective quantification of over 337 distinct lipids from over 28 lipid subclasses, as previously described (41). Individual lipid species were measured by multiple reaction monitoring transitions.
Ion Mode:NEGATIVE
  
MS ID:MS002745
Analysis ID:AN002955
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Lipid profiling was performed using an Agilent 1260 HPLC coupled to an Agilent 6490 triple quadrupole (QQQ) mass spectrometer (41). Each sample was run through three separate chromatographic conditions (reverse-phase negative mode, reverse-phase positive mode and normal-phase positive mode) for the effective quantification of over 337 distinct lipids from over 28 lipid subclasses, as previously described (41). Individual lipid species were measured by multiple reaction monitoring transitions.
Ion Mode:POSITIVE
  
MS ID:MS002746
Analysis ID:AN002956
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Lipid profiling was performed using an Agilent 1260 HPLC coupled to an Agilent 6490 triple quadrupole (QQQ) mass spectrometer (41). Each sample was run through three separate chromatographic conditions (reverse-phase negative mode, reverse-phase positive mode and normal-phase positive mode) for the effective quantification of over 337 distinct lipids from over 28 lipid subclasses, as previously described (41). Individual lipid species were measured by multiple reaction monitoring transitions.
Ion Mode:POSITIVE
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