Development of high-throughput flow cytometry for synaptosomes
Date Issued
2015
Date
2015
Author(s)
Chen, Hui-Ting
Abstract
Alzheimer’s disease (AD) is the most common type of dementia, a protein misfolding disorder of beta amyloid and tau, and a neurodegenerative disease characterized by the loss of neurons and synapses. Loss of synaptic terminals in AD brains exhibits stronger correlations with decreased cognitive function than cell death. Disruption of synaptic function is a key event in early cognitive decline. Consequently, neuroscientists used enriched preparation of synaptosomes to study synapse dysfunction and try to find its relationship with the disease. Flow cytometry has been applied to the study of synapses by several research groups. However, the immunostaining procedure of synaptosomes prior to flow cytometry represents a major technical obstacle. Conventional immunostaining of synaptosomes for flow cytometry involves many sedimentation steps which lead to cumulative damage of these fragile organelles, resulting in significant sample loss and signal variability. We devised a method to circumvent this issue by modifying synaptic surfaces with desthiobiotin, which can be captured in wells with streptavidin-coated bottoms. Surfaced-attached synaptosomes can be immunostained under gentle conditions and imaged with inverted microscopes. Elution with biotin releases synaptic terminals from the bottom surface for flow cytometry analysis. This capture-and-release strategy enables high-throughput analysis of brain synapses with minimal tissue samples. So far, we tried three different surface modification reagents. First, we tried Sulfo-NHS-SS-Biotin(Sulfo-N-hydroxysuccinimide-Disulfide-Biotin). However, this led to poor immunostaning because the reducing agents used to cleave the disulfide bond and release the synaptosomes will also cleave the disulfide bonds of antibodies. As a result it was not suitable for modifying synaptosomes. The second modifying reagent we used was NHS-Desthiobiotin(N-hydroxysuccinimide-Desthiobiotin), as the releasing process could be accomplished by competitive elution with biotin. However, this reagent was also found to be unsuitable. It might have modified both the surface of synaptosomes and the epitopes inside the synaptosomes due to its membrane permeability, leading to poor immunostaining. The last one we used was Sulfo-NHS-LC-Desthiobiotin(Sulfo-N-hydroxysuccinimide-Long Chain-Desthiobiotin), which contained a sulfite group and thus was impermeable to cell membrane. This produced higher levels of immunostaining of labeled synaptosomes captured in wells compared to the unlabeled control group. However, the amount of captured synaptosomes was still not satisfying. This may be due to comparatively low binding affinity between desthiobiotin and streptavidin, which is only 1/1000 of that between biotin and streptavidin, or due to the detachment of streptavidin from the plate surface, or perhaps the short linker was sub-optimal for capturing. To sum up, this capture-and-release strategy still requires further optimization.
Subjects
Alzheimer’s disease
synaptosome
streptavidin
biotin
desthiobiotin
flow cytometry
Type
thesis
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