Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry
Published: February 29, 2024
Abstract
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.
Protocol
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 Sterilize a 33 G needle and syringe: disassemble and autoclave at 115 οC. Prepare needles for injection by rising in sterile phosphate-buffered saline (PBS). Thaw fluorescently labeled particles at room temperature for 5 – 10 min. Prepare partic…
Representative Results
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…
Divulgations
The authors have nothing to disclose.
Materials
| 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 |
Tags
Citer Cet Article
Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry. J. Vis. Exp. (Pending Publication), e21986, doi: (2024).