Source: Murinello, S. et al., Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay. J. Vis. Exp. (2016)
This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Materials for Injection
2. Intravitreal Injection of Bead Solution
NOTE: Two people are required to perform the injection, in a way such that the person performing the injection can hold the mouse and maintain the focus on the eyeball, while the other person passes the loaded syringe and pushes the plunger.
3. Harvesting of Retinal Tissue
NOTE: Retinal tissue from eyes not injected with fluorescently labeled particles should be collected as a control for flow cytometric analysis. Though the assay can be performed using a single retina, for best performance, two retinas should be pooled together.
4. Preparing a Single Cell Suspension
5. Staining Single Cell Suspensions for Flow Cytometric Analysis
6. Flow Cytometric Analysis
Figure 1: Intravitreal Injection Setup and Representative Retinal Section showing Fluorescently Labeled Particles in Retinal Layers. (A) A gel pack is positioned under the dissection microscope. (B) A rodent is held as shown to position the eye for intravitreal injection. (C) Three hours after injection fluorescently labeled particles can be seen throughout most retinal layers. (D, E) CX3CR1GFP/GFP mice and particles labeled with a red fluorophore were used to visualize particle uptake by microglia. Microglia in the deeper (D) and superficial (E) layers take up particles. Scale bars – 20 µm.
Figure 2: Gating Strategy to Select Phagocytic Microglia. (A) Single-cell suspensions from retinal tissue from a p10 mouse are analyzed. (B) After excluding dead cells (PI+), (C) CD11b+ cells are selected. (D) To select only for microglia, CD11b+ neutrophils (Ly6G+) and monocytes (Ly6C+) are excluded. (E) Phagocytic microglia will have taken up fluorescently labeled particles. (F) The number of phagocytic CD11b+ cells is similar to that of microglia (CD11b+Ly6G–Ly6C–). n= 9, pooled from three independent experiments; error bars represent mean ± SEM.
The authors have nothing to disclose.
Stereomicroscope | Nikon | Discontinued | |
Hamilton syringe, 600 series | Sigma | 26702 | |
33 gauge, Small Hub RN NDL, 0.5 in, point style 4 – 12o | Hamilton | 7803-05 | |
Zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate | ThermoFisher Scientific | Z-23373 | Prepare immediately before injection |
DPBS | Corning | 21-030-CV | |
Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | Need two |
Dumont #5SF Forceps | Fine Science Tools | 11252-00 | |
Vannas Spring Scissors – 3mm Cutting Edge | Fine Science Tools | 15000-10 | Curved |
Neural Tissue Dissociation Kit – Postnatal Neurons | Miltenyi Biotec | 130-094-802 | |
5 mL Polystyrene Round-bottom Tube | Falcon | 352054 | |
96 well U-bottom plate | Falcon | 353077 | |
Stain Buffer (BSA) | BD Biosciences | 554657 | |
CD11b-BV650 Antibody | BioLegend | 101259 | |
Ly6C-APC-Cy7 | BioLegend | 128025 | |
Ly6G-PE-Cy7 | BioLegend | 127617 | |
Propidium Iodide | BD Biosciences | 556463 | |
Purified anti-mouse CD16/32 Antibody | BioLegend | 101301 |