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…

Declarações

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

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Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry. J. Vis. Exp. (Pending Publication), e21986, doi: (2024).

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