Stem Cell Research Source

Analysis of hESC-derived self-renewing neural stem cells (NSCs) by bioimaging and flow cytometric analysis.

A Time line for differentiating human embryonic stem cells (hESCs) into self-renewing neural stem cells.

B Human embryonic stem cell-derived neural stem cells were cultured on BD Falcon™ 96 well imaging plates (Cat. No. 353219) that were coated with poly-L-ornithine and laminin. Cells were fixed with 4% paraformaldehyde, followed by BD Perm/Wash buffer (Cat. No. 554723). Multicolor cell staining was performed

with the following antibodies: Ki-67 Alexa Fluor® 488 (Cat. No. 558616) pseudo-colored yellow, Sox2 Alexa Fluor® 647 (Cat. No. 560294) pseudo-colored green, and Nestin Alexa Fluor® 555 pseudo-colored red.

Cell nuclei were counterstained using Hoechst 33342 pseudo-colored blue. The cells were imaged on a BD Pathway™ 435 bioimager using a 20x objective.

C-D Neural stem cells were dissociated and fixed using BD Cytofix/Cytoperm™ fixation/permeabilization solution (Cat No. 554722), then washed using BD Perm/Wash buffer. Cells were stained

with Sox2 PE (Cat. No. 560291), Nestin Alexa Fluor® 647, and OCT3/4 Alexa Fluor® 488 (Cat. No. 560253) antibodies. Purity of neural stem cells is approximately 99% co-expressing Nestin and Sox2.

E Samples were prepared as described above. Cells were stained with Ki-67 Alexa Fluor® 488 and Sox2 Alexa Fluor® 647 and then run on a BD FACSCanto™ II cytometer and analyzed with BD FACSDiva software.

F Key to markers and cell types recognized.