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CHI SUN, Deborah L Stenkamp; Isolation of photoreceptors from mature, developing, and regenerating zebrafish retinae, and of microglia from regenerating zebrafish retinae. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3107. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
This work describes experimental procedures for the dissociation of retina of the zebrafish (Danio rerio) to produce a suspension of single cells, and for subsequent fluorescence-activated cell sorting (FACS). Establishing this methodology to purify photoreceptors and retinal microglia is important for the identification of key molecular signatures that underlie their functions in development, survival and/or regeneration.
Specific transgenic zebrafish lines were in this research, such that in each line, a subtype of photoreceptors or microglia expresses a type of designated fluorescent protein. Three sorting characteristics were utilized for FACS, namely, compensation analysis, forward side scatter (FSC) and fluorescence intensity. RNA samples were isolated from sorted samples for analysis of RNA quality by bioanalyzer or fragment analyzer, and for analysis of gene expression. Sorting purity was validated by post-sorting analysis and/or by qRT-PCR detection of cell-specific genes.
Methods for dissociation of zebrafish retinae, FACS, and RNA isolation were optimized. This methodology has been applied to isolate pure sorted samples of rods and long wavelength-sensitive (LWS) cones from developing retinae at 14 and 30 days post-fertilization (dpf); rods and LWS cones from 2-3 regenerating retinae at 14 and 30 days post-injury (dpi); rods, LWS cones, medium wavelength-sensitive (MWS; Rh2-2) cones, short wavelength-sensitive (SWS2) cones, and UV-sensitive cones from 2-4 adult mature retinae. We also successfully separated lws1-expressing LWS cones from lws2-expressing LWS cones from 4 fish of a transgenic line in which lws1 is reported with green fluorescence protein (GFP) and lws2 is reported with red fluorescence protein (RFP). On average, 9000 activated microglia were sorted from 8 regenerating (7 dpi) retinae of a transgenic line in which microglia express GFP. Electropherograms verified downstream isolation of high-quality RNA from sorted samples. Examples of post-sorting analysis, as well as results of qRT-PCR studies validated the good sorting purity.
We successfully isolated specific photoreceptor subtypes from developing, regenerating, and mature retinae, and microglia from regenerating retinae. Highly pure sorted samples can subsequently be used for gene expression analysis, such as qRT-PCR and RNA-seq.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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