Xfe24 extracellular flux analyzer

Mitochondrial function was determined with an XFe24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, United States). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in dechorionated embryos at 48 hpf. Embryos were staged and placed one per well on an islet capture microplate filled with E3 egg water. The plate was incubated in an incubator without CO₂ at 28°C for 30min. After measuring baseline OCR as an indication for basal respiration, OCR was measured after an injection of 2μM of FCCP to determine maximal respiration.

The up-regulated AD-specific cytokine profile of IFNγ, IP-10/CXCL10, and IL-9, as determined by the multivariate modeling, was applied to primary neuron and astrocyte cultures derived from CD1 P0 neonates. The combination cytokine treatment was applied 72 h prior to experimentation in levels proportional to the concentrations we measured in 5xFAD 180-day hippocampus samples. Concentrations were centered in the nanomolar range previously used to study acute cytokine responses in neuron cultures [27 (link), 99 (link)]. Recombinant murine cytokines were purchased from Peprotech, IFN-γ (cat 315-05), IP-10/CXCL10 (cat 250-16), and IL-9 (cat 219-19), and reconstituted to 1 mg/mL in sterile water and diluted to 250 μg/mL (IL-9) or 10 μg/mL (IFNγ and IP-10/CXCL10) in 0.1% cell-culture grade bovine serum albumin (Sigma A9418) in 1X PBS. 5 nM IFNγ, 12 nM IP-10/CXCL10, and 500 nM IL-9 or an equal amount of 0.1% bovine serum albumin vehicle were added to the appropriate neuronal or glial cell culture medium for treatment. Neuron cultures were treated 9 or 10 days after plating, and astrocytes were treated, following the removal of non-adherent cells, at 50% confluency. After a 72-h stimulation, cells were assayed on the Seahorse XFe24 Extracellular Flux Analyzer or lysed for RNA isolation.

PC12 cells (2X10⁴ cells/well) were seeded in 24 well Seahorse XF24 Cell Culture Microplates and transfected with Con-siRNA or Ahi1-siRNA for 48 h. Cells were washed with ATP Assay Medium (Agilent XF DMEM Medium pH 7.4; 10 mM XF Glucose; 1 mM XF Sodium Pyruvate; 2 mM XF L-Glutamine) and incubated with warm ATP Assay Medium at 37 °C (non-CO₂) for 45 min. The medium was removed and replaced with fresh ATP Assay medium. Then, the plates were placed on the Seahorse XFe24 Extracellular Flux Analyzer. The ATP production rate was measured under stress conditions in response to 1.5 μM of oligomycin and 0.5 μM rotenone plus antimycin A according to the manufacturer’s instructions (Agilent 103,592–100). The ATP production rate was normalized to the protein concentrations. Data were analyzed in the Agilent ATP Assay Report Generator, and statistical analyses were performed using GraphPad Prism 8.0.