Stem Cell Research Source

Enrichment of hESC-derived neurons by sorting with the BD FACSAria II cell sorter.

A hESC-derived neural stem cells were differentiated for one week in the presence of BDNF, GDNF, and dibutyrl-cyclicAMP. Cells were cultured in both BD Falcon™ 96-well imaging plates (Cat. No. 353219) and 10-cm plates coated with poly-L-ornithine and laminin. Cells were fixed in 4% paraformaldehyde followed by BD Perm/Wash Buffer (Cat. No. 554723) and imaged with the following fluorochrome-conjugated

antibodies: neural stem cell markers Sox2 Alexa Fluor® 647 (Cat. No. 560294) pseudo-colored yellow and Nestin Alexa Fluor® 555 pseudo-colored red, and neuronal marker Map2b Alexa Fluor® 488 pseudo-colored green. Cell nuclei were counterstained with Hoechst 33342 pseudo-colored blue. Staining indicated a heterogeneous population of cells prior to sorting. The cells were imaged on a BD Pathway™ 435 bioimager using a 10x objective.

B Cultured cells (from 10-cm plates as in A) were dissociated, and then cells were sorted on a BD FACSAria™ II sorter at 70 psi using a 70-µm nozzle using antibodies to CD markers. The sorted cells were plated on a BD Falcon 96 well imaging plate, then fixed and stained as above. The staining patterns demonstrated that sorting resulted in enrichment of neurons (Map2b-positive cells).

C Key to markers and cell types recognized.

Cell surface marker analysis of hESC-derived neural stem cells using flow cytometry.

A Unstained cells and gating strategy.

B Neural progenitors were stained with anti-CD24 PE (Cat. No. 555428) and SSEA-1 FITC (Cat. No. 560127). Nearly 100% of the neural progenitors stained positive for CD24, while 77% of the neural progenitors stained positive for SSEA-1.

C Neural progenitors were stained with anti-CD95 PE (Cat. No. 555674) and SSEA-1 FITC. Of the CD95-positive cells, nearly all were SSEA-1-positive. Cells were run on a BD FACSAria II sorter and analyzed with BD FACSDiva™ software.