Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

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…

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