Brains were rapidly isolated from HIV-1 Tg and litter mate control animals. The cerebellum was removed. After extraction, brains were immediately rinsed with ice-cold PBS to remove blood. The meninges were removed. Tissue was chopped and homogenized using a Dounce homogenizer. Synaptic mitochondria were isolated using a synaptosomal mitochondria preparation as previously performed [30 (link)–32 (link)]. Mitochondria for mass spectrometry were lysed in 4% sodium dodecyl sulfate (SDS), and protein concentration was quantified using a using a Pierce 660 assay with bovine serum albumin standards (Thermo Fisher Scientific, Rockford, IL). Mitochondria for functional analysis were placed in a MAS buffer and treated according to manufacturers’ protocols for the experiments.
Protocol detail
Isolation of Synaptic Mitochondria from HIV-1 Tg Brains
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Animals
Assay
Blood
Bovine serum albumin
Brains
Buffer
Cerebellum
Cold
Hiv 1
Mass spectrometry
Meninges
Mitochondria
Protein
Sodium dodecyl sulfate
Synaptosomal
Tissue
Corresponding Organization :
Other organizations : University of Nebraska Medical Center
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Variable analysis
independent variables
- HIV-1 Tg animal vs. litter mate control animals
- Synaptic mitochondria properties
- Mitochondrial function
- Cerebellum removal
- Meninges removal
- Tissue chopping and homogenization
- Synaptosomal mitochondria preparation
- Mitochondria lysis in 4% sodium dodecyl sulfate (SDS)
- Protein concentration quantification using Pierce 660 assay
- Mitochondria treatment in MAS buffer according to manufacturer's protocols
- Positive control: Not specified
- Negative control: Litter mate control animals
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