A Protocol for FRET-Based Live-Cell Imaging in Microglia

Abstract

Summary This protocol highlights the use of FRET-based biosensors to investigate signaling events during microglia activation in real time. Understanding microglia activation has gained momentum as it can help decipher signaling mechanisms underlying the neurodegenerative process occurring in neurological disorders. Unlike more traditional methods widely employed in the microglia field, FRET allows microglia signaling events to be studied in real time with exquisite subcellular resolution. However, FRET-based live-cell imaging requires application-specific biosensors and specialized imaging systems, limiting its use in in vivo studies. For complete details on the use and execution of this protocol, please refer to Socodato et al. (2020) , Portugal et al. (2017) , and Socodato et al. (2018) .

Astrocyte-derived TNF and glutamate critically modulate microglia reactivity by methamphetamine

“…Primary microglia or astrocyte were plated on plastic-bottom culture dishes μ-Dish35mm (iBidi, Martinsried, DE) and transfected with FRET biosensor for glutamate (pDisplay FLIPE-600n, plasmid 13545), ROS (pFRET-HSP33 cys, plasmid 16076) or calcium (pcDNA-D1ER, plasmid 36325), all from Addgene (MA, USA) using jetPRIME ® from Polyplus (NY, USA). Imaging was performed using a Leica DMI6000B inverted microscope, and images were processed in FIJI software exactly as before 36 .…”

Rac1 signaling in microglia is essential for synaptic proteome plasticity and experience-dependent cognitive performance

“…The human microglial cell line HMC3 was obtained from ATCC (ATCC ® CRL-3304 ™ ). Cells were cultivated and maintained as before (4,37,50). HMC3 microglia were plated on plastic-bottom culture dishes (µ-Dish 35 mm, iBidi) and transfected, as before (50), with different biosensors.…”

“…The native 32-bit values from ratio images (GDNF over mCherry or YFP) were used for statistics. Ratio images are shown in intensity-modulated display (50). Statistical analyses were conducted using unpaired t-test (single cells were considered the experimental units; statistical significance was defined as P < 0.05).…”

“…The mean gray intensity values from spine GCaMP6f and spine actin dynamics (lifeact fluorescence signal) were normalized using the ΔF/F. Representative GCaMP6f images are shown in intensity-modulated display (50).…”

“…The mean gray intensity values from ratio images were normalized using the deltaF/F0 for statistics. Representative images are shown in intensity-modulated display (50). Statistical analyses were conducted using repeated measures two-way ANOVA (individual microglial cells were considered the experimental units; statistical significance was defined as P < 0.05) with the Greenhouse-Geisser correction (to combat sphericity violation) and the Šidák correction for multiple comparisons.…”