Journal: PLoS ONE

Article Title: A standardized human embryoid body platform for the detection and analysis of teratogens

doi: 10.1371/journal.pone.0171101

Figure Lengend Snippet: (A) Flow cytometric analysis of permeablized H9 hESCs. Cells were exposed to antibodies against TRA1-60, SSEA4, NANOG and OCT4. IgG antibody was used as negative control. Top: Cytometry profiles of undifferentiated H9 cells. Bottom: Cytometry histograms of H9 cells 9 days after removal of bFGF. (B) Flow cytometric analysis of undifferentiated H9 cells for the co-stained for TRA1-60 and OCT4. (C) Quantification of positive cells for TRA1-60, SSEA4, NANOG and OCT4 in comparison to IgG control for both undifferentiated and differentiated H9 cells.

Article Snippet: Following cell fixation and permeabilization, the cells were incubated with primary antibodies for 30 minutes on ice using Stain buffer (BD Biosciences, #554657): Mouse α-human Tra1-60-FITC (BD Biosciences, #560380), Mouse α-human SSEA4 PE (BD Biosciences, #560128), Mouse α-human Oct3/4 AlexaFluor 647 (BD Biosciences, #560329), Mouse α-human Nanog-PerCP-Cy5.5 (BD Biosciences, #562259).

Techniques: Negative Control, Cytometry, Staining

Journal: PLoS ONE

Article Title: A standardized human embryoid body platform for the detection and analysis of teratogens

doi: 10.1371/journal.pone.0171101

Figure Lengend Snippet: (A) Preparation of spheroids from TRA1-60+ magnetically purified hESCs. Low passage H9 cells were dissociated to single cells by Accutase treatment, then TRA1-60+ cells were magnetically purified. These were then plated at known density into Aggrewell plates to from spheroids of uniform size. (B) Alternative plating and differentiation approaches. Three methods were assessed: (1) Spheroids were deposited individually into cell culture plate wells, and imaged every 3 days. (2). Pools of EBs are plated into 6 well dishes under non-adherent conditions, imaged, quantitated for size. (3) Spheroids are attached onto Matrigel coated plates and allowed to proliferate and spread under adherent conditions.

Article Snippet: Following cell fixation and permeabilization, the cells were incubated with primary antibodies for 30 minutes on ice using Stain buffer (BD Biosciences, #554657): Mouse α-human Tra1-60-FITC (BD Biosciences, #560380), Mouse α-human SSEA4 PE (BD Biosciences, #560128), Mouse α-human Oct3/4 AlexaFluor 647 (BD Biosciences, #560329), Mouse α-human Nanog-PerCP-Cy5.5 (BD Biosciences, #562259).

Techniques: Purification, Cell Culture

Journal: PLoS ONE

Article Title: A standardized human embryoid body platform for the detection and analysis of teratogens

doi: 10.1371/journal.pone.0171101

Figure Lengend Snippet: Spheroids containing uniform numbers of cells were prepared in Aggrewell plates using TRA1-60 + cells. (A) Aggrewell plate containing spheroids aggregated for 3 days (1.25X magnification). (B) Aggrewell plate containing spheroids aggregated for 3 days (4X magnification). (C) Aggrewell plate containing spheroids aggregated for 3 days (10 X magnification). (D) Spheroids imaged immediately after aggregation.

Article Snippet: Following cell fixation and permeabilization, the cells were incubated with primary antibodies for 30 minutes on ice using Stain buffer (BD Biosciences, #554657): Mouse α-human Tra1-60-FITC (BD Biosciences, #560380), Mouse α-human SSEA4 PE (BD Biosciences, #560128), Mouse α-human Oct3/4 AlexaFluor 647 (BD Biosciences, #560329), Mouse α-human Nanog-PerCP-Cy5.5 (BD Biosciences, #562259).

Techniques: