April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Evaluation of Human Y 79 Cell Lines for Putative Stem Cell Properties by Single Cell Assay and Gene Expression
Author Affiliations & Notes
  • M. S. Balla
    Kallam Anji Reddy Campus,
    LV Prasad Eye Institute, Hyderabad, India
  • G. K. Vemuganti
    Kallam Anji Reddy Campus,
    LV Prasad Eye Institute, Hyderabad, India
  • C. Kannabiran
    Kallam Anji Reddy Molecular Genetics Lab,
    LV Prasad Eye Institute, Hyderabad, India
  • S. G. Honavar
    Kallam Anji Reddy Campus,
    LV Prasad Eye Institute, Hyderabad, India
  • Footnotes
    Commercial Relationships  M.S. Balla, None; G.K. Vemuganti, None; C. Kannabiran, None; S.G. Honavar, None.
  • Footnotes
    Support  ICMR, C-TRACER, Hyderabad Eye Research Foundation, CSIR
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2643. doi:
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      M. S. Balla, G. K. Vemuganti, C. Kannabiran, S. G. Honavar; Evaluation of Human Y 79 Cell Lines for Putative Stem Cell Properties by Single Cell Assay and Gene Expression. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2643.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Cells from tumors like retinoblastoma are suspected to have two kinds of cell populations, one being quiescent stem like cells, and another dividing cells. In order to investigate and quantify the populations, we studied the human Rb cell line Y79 for their clone forming ability, differential gene expression and cell cycle status.

Methods: : Y79 cell line was maintained in RPMI with 10% FCS. Single cell assay was performed to evaluate clone-forming ability. One million cells were stained and analyzed for CD133 by Flow cytometry and sorted for CD133+/CD133- populations. These two subpopulations were then evaluated for cell cycle status by propidium iodide labeling, and differential expression of putative stem/progenitor cell markers ABCG2, PROX1 by RT-PCR.

Results: : Clone forming ability was noted in 26.6±3.8% cells and CD133 expression in 79.7±1.3% of the cells. RT-PCR analysis of the CD133- population showed the expression of PROX1, which was not detected in the CD133+ cells. Majority of CD133- population (83.3±4.1%) were in G0/G1 phase, while CD133+ cells were predominantly (81.1±10.6%) in S, G2/M phase as assessed by PI staining.

Conclusions: : The Y79 cell line showed presence of cells with clone- forming ability and differential expression of CD133, thus supporting the existence of putative stem-like cells. Expression of PROX1 and quiescence of CD133- cells, further substantiate this hypothesis.

Keywords: flow cytometry • retinoblastoma • gene/expression 
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