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…
Disclosures
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