NOVEL ISOLATION OF CHICKEN RETINAL HORIZONTAL CELLS

MORERA L P; DIAZ, N.; GUIDO M

San Luis

Encuentro; XLVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

Sociedad argentina de investigacion en bioquimica y biologia molecular

We described that the non-visual photopigment melanopsin X is expressed mainly in horizontal cells of the chicken retina (Verra et al., 2011). In this work our aim was to purify and culture horizontal cells (HC´s) from the chicken embryonic retina for further characterization. Disaggregated retinas of chicken embryos at day 14 were subjected to a bovine serum albumin (BSA) discontinuous gradient of concentrations ranging from 1 to 5%. After centrifugation, cells collected from the different phases were cultured for 4 days and characterized by immunochemistry and cell morphology. Phases were examined with specifics antibodies against HC markers: PROX-1 and Islet-1. Results showed that only the fraction corresponding to 2.5% of BSA was highly enriched in PROX-1 positive cells (> 95%) displaying a typical HC morphology. In fact, some cells in this fraction resembled axon-less candelabrum-shaped HC’s. Preliminary results showed that around 50% of the PROX-1 cells in this phase were positive for Islet-1. Moreover, Western blot assays demonstrated that mainly the phase at 2.5% BSA, exhibited positive PROX-1 immunoreactivity (MW: 83kDa). In conclusion the BSA gradient proved to be the most effective method to separate different retinal cell populations, and particularly HC´s which will allow us to characterize them as potential photoreceptors by biochemical and pharmacological studies.