Isolation and Culture of Retinal Ganglion Cells

Retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning protocol (Dvoriantchikova et al., 2011 (link); Dvoriantchikova et al., 2012 (link)). Briefly, the whole retinas were incubated in papain solution (16.5 U/mL) for 30 minutes and then macrophage and endothelial cells were removed from the cell suspension by panning with the anti-macrophage antiserum (Accurate Chemical, Westbury, NY). In the next step, RGCs were bound to the panning plates containing anti-Thy1.2 antibody and released by trypsin incubation. Primary RGCs were cultured in Neurobasal/B27 media (Life Technologies, Grand Island, NY) one day before the experiment. Astrocytes and microglial cells were prepared from the brains of neonatal (postnatal day 3) mice as previously described (Dvoriantchikova et al., 2011 (link); Barakat et al., 2012 (link); Santos et al., 2014 (link)). These cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Grand Island, NY) and 1% antibiotic/antimycotic (Life Technologies, Grand Island, NY).

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Dvoriantchikova G., Santos A.R., Danek D., Dvoriantchikova X, & Ivanov D. (2014). The TIR-domain-containing adapter inducing interferon-β-dependent signaling cascade plays a crucial role in ischemia–reperfusion-induced retinal injury, whereas the contribution of the myeloid differentiation primary response 88-dependent signaling cascade is not as pivotal. The European Journal of Neuroscience, 40(3), 2502-2512.

Publication 2014

Neurobasal/B27 media Dulbecco’s Modified Eagle’s Medium (DMEM) fetal bovine serum (FBS) antibiotic/antimycotic

Anti thy1 2 antibody Antibiotic Antiserum Astrocytes Brains Cells Cultured medium Eagle Endothelial cells Fetal bovine serum Macrophage Mice Microglial cells Neonatal Papain Retinal ganglion cells Retinas Trypsin

Corresponding Organization : University of Miami

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Isolation of retinal ganglion cells (RGCs) using the two-step immunopanning protocol
Preparation of astrocytes and microglial cells from the brains of neonatal (postnatal day 3) mice

dependent variables

Not explicitly mentioned

control variables

Papain solution concentration (16.5 U/mL) and incubation time (30 minutes) for retinal digestion
Culture media used for RGCs (Neurobasal/B27) and astrocytes/microglial cells (DMEM with 10% FBS and 1% antibiotic/antimycotic)

controls

Positive control: Anti-Thy1.2 antibody used to bind and isolate RGCs
Negative control: Anti-macrophage antiserum used to remove macrophage and endothelial cells from the cell suspension prior to RGC isolation

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