Retinas were homogenized and pooled as previously described (Williams et al., 2017a (link)). Eyes were enucleated and placed directly into ice-cold HBSS. Retinas were dissected and placed into 100uL of HBSS with dispase (5U/mL) DNAse 1 (2000U/mL). Retinas were incubated for 20 minutes at 37°C whilst being shaken at 350RPM in an Eppendorf Thermomixer R. Samples were gently triturated. For flow cytometric analysis, each sample was stained with DAPI, CD45 BV605 (clone 30-F11, BD Biosciences, 1:240), and CD11b PE (clone M1/70, Biolegend, 1:960), followed by processing on a FACSymphony A5 cytometer, and analysis using FlowJo (v10) software.
Protocol detail
Retina Immune Cell Isolation and Analysis
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Corresponding Organization : University of Rochester Medical Center
Other organizations : Tufts University
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Variable analysis
independent variables
- Dispase concentration (5U/mL)
- DNAse 1 concentration (2000U/mL)
- Incubation time (20 minutes)
- Shaking speed (350RPM)
- Percentage of CD45+ and CD11b+ cells in the retinal cell population (measured by flow cytometry)
- Temperature (37°C)
- Cell dissociation protocol (homogenization, trituration)
- Flow cytometry staining (DAPI, CD45, CD11b)
- Flow cytometry instrument (FACSymphony A5)
- No positive or negative controls were explicitly mentioned in the input protocol.
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