Immunophenotyping of Pluripotent and Neural Stem Cells

Samples were prepared as described previously (Al‐Attar et al., 2018 (link)). Briefly, harvested cells were fixed with 2% paraformaldehyde, washed with permeabilization buffer (BioLegend) and incubated with primary antibodies: Oct4 (Chemicon), Nanog (Cell Signaling), Sox2 (Santa Cruz Biotechnology), Nestin (Novus Biologicals), and Rabbit IgG (Abcam) and chicken IgY isotype (R&D Systems) controls. After rinsing, cells were incubated with an Alexa flour‐488 goat anti‐rabbit secondary antibody (Molecular Probes), or a FITC goat anti‐chicken IgY (Novus Biologicals) isotype control. Stained samples were read on an LSR‐II cytometer using FACSDiva version 6 (BD Biosciences) and analyzed by FlowJo Software (TreeStar; version 9.5). Data are represented as geometric mean‐fluorescence intensity (GMFI).

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Bazina F., Brouxhon S.M, & Kyrkanides S. (2021). Reprogramming oral epithelial keratinocytes into a pluripotent phenotype for tissue regeneration. Clinical and Experimental Dental Research, 7(6), 1112-1121.

Publication 2021

permeabilization buffer Nanog Nestin FACSDiva

Anti antibody Antibodies Biologicals Buffer Cells Chicken Fitc Flour Fluorescence Goat Isotype Molecular probes Nestin Novus Oct4 Paraformaldehyde Rabbit Sox2

Corresponding Organization : University of Kentucky

Other organizations : Stony Brook School, Stony Brook University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Geometric mean-fluorescence intensity (GMFI)

control variables

Fixed cells with 2% paraformaldehyde
Washed with permeabilization buffer (BioLegend)
Primary antibodies: Oct4 (Chemicon), Nanog (Cell Signaling), Sox2 (Santa Cruz Biotechnology), Nestin (Novus Biologicals)
Rabbit IgG (Abcam) and chicken IgY isotype (R&D Systems) controls
Alexa flour-488 goat anti-rabbit secondary antibody (Molecular Probes)
FITC goat anti-chicken IgY (Novus Biologicals) isotype control
LSR-II cytometer using FACSDiva version 6 (BD Biosciences)
FlowJo Software (TreeStar; version 9.5)

positive controls

Rabbit IgG (Abcam)
Chicken IgY isotype (R&D Systems)

negative controls

Rabbit IgG (Abcam)
Chicken IgY isotype (R&D Systems)

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