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MB Sample ID: SA193731

Local Sample ID:42 - ICD 10
Subject ID:SU002137
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

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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.