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MB Sample ID: SA193766

Local Sample ID:15 - CA1 15
Subject ID:SU002137
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

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Subject:

Subject ID:SU002137
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
15 - CA1 15SA193766FL023733Lung Progenitor CellDifferentiation stage

Collection:

Collection ID:CO002130
Collection Summary:Samples were analyzed for 180 metabolites using the AbsoluteIDQ® p180 kit from Biocrates Life Sciences AG at the Analytical Facility for Bioactive Molecules (The Hospital for Sick Children, Toronto, Canada). Collected cell pellets were resuspended in 15 % ice-cold 10 mM Phosphate Buffer (PB) + 85 % EtOH as per AbsoluteIDQ® p180 kit protocol. Resuspended cells were subjected to three rounds of sonication and rapid freeze thawing. Final suspension was then centrifuged at 20,000 x g for 10 minutes at 4°C. A small aliquot was taken for protein assay, and the remainder of the supernatant was used for metabolic analysis. Samples, standards, and controls (10 mL each) were added to the Biocrates 96-well filter plate with internal standard, dried under nitrogen, derivatized with phenyl isothiocyanate, and then extracted with methanol, as per kit instructions.
Sample Type:human cells

Treatment:

Treatment ID:TR002149
Treatment Summary:No treatment

Sample Preparation:

Sampleprep ID:SP002143
Sampleprep Summary:At each differentiation step, DE, AFE and LPC, cells were analyzed by FACS for stage specific markers. The differentiated cells were harvested with TrypLE at 37 °C for 5-10 minutes and collected by centrifugation at 300 g for 5 minutes at room temperature. The cell pellets were resuspended in 3% (v/v) FBS in PBS, cell aggregates were removed using a cell strainer with a 40-µm pore size and single cells were collected. For surface antigens, the cells were incubated with primary antibodies for 30 minutes on ice with gentle shaking, washed twice with 3% (v/v) FBS in PBS, and if necessary, incubated with the secondary antibodies for 30 minutes followed by two more rinses with 3% (v/v) FBS in PBS. For DE cells, we used PE conjugated mouse anti-human CXCR4 and APC conjugated mouse anti-human cKit antibodies; AFE cells were stained with PE-conjugated mouse anti-human CD56 and APC conjugated mouse anti-human CD271 antibodies and LPCs we identified using mouse anti-human CPM antibody. Alexa Fluor 488 donkey anti-mouse IgG was used as the secondary antibody against primary CPM antibody. The cells were either sorted using the BD aria FACS sorter at the Sick Kids FACS core and/or analyzed using a Beckman Coulter Gallios flow cytometer. Unstained controls were used to set up gates for FSC and SSC and double stained cells underwent fluorescence minus one (FMO) to ensure accurate gating. Sorted cells were collected and the spun down into a cell pellet and stored in -80C degrees for further processing. The endodermal differentiations and cell sorts were repeated at four different times with two biological repeats.

Combined analysis:

Analysis ID AN003346 AN003347
Analysis type MS MS
Chromatography type Reversed phase None (Direct infusion)
Chromatography system Agilent 1290 Infinity -
Column Agilent Eclipse XDB-C18 (100 x 3.0mm) -
MS Type ESI ESI
MS instrument type Triple quadrupole Triple quadrupole
MS instrument name ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap
Ion Mode POSITIVE UNSPECIFIED
Units uM uM

Chromatography:

Chromatography ID:CH002477
Instrument Name:Agilent 1290 Infinity
Column Name:Agilent Eclipse XDB-C18 (100 x 3.0mm)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002478
Instrument Name:-
Column Name:-
Chromatography Type:None (Direct infusion)

MS:

MS ID:MS003115
Analysis ID:AN003346
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Analyst1.7 (Sciex); MetIDQ (Biocrates)
Ion Mode:POSITIVE
  
MS ID:MS003116
Analysis ID:AN003347
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Analyst1.7 (Sciex); MetIDQ (Biocrates)
Ion Mode:UNSPECIFIED
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