The rate of H2O2 production in mitochondria was determined using the oxidation of the fluorogenic indicator amplex red in the presence of HRP as we reported before.47 (link) The concentrations of HRP and amplex red in the incubation were 0.1 unit/ml and 50 μM, respectively. Fluorescence was recorded in a microplate reader (1420 Victor2, PerkinElmer Life Sciences) with 530 nm excitation and 590 nm emission wavelengths. Standard curves obtained by adding known amounts of H2O2 to assay medium in the presence of the reactants (amplex red and HRP) were linear up to 2 μM. Isolated mitochondria were incubated at 0.1 mg of protein/ml at 30 °C with compound for 1 h. H2O2 production was initiated in mitochondria using glutamate (10 mM) + malate (2.5 mM), or succinate (5 mM) as substrates. Rotenone (2.4 μM) was added into incubation medium to inhibit the activities of complex I when succinate was used as complex II substrate. The rate of H2O2 production was linear with respect to mg of mitochondrial protein.
1420 victor 2
Manufactured by PerkinElmer
Sourced in Italy
The 1420 Victor 2 is a multimode microplate reader designed for life science research applications. It is capable of performing absorbance, fluorescence, and luminescence measurements on sample plates.
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Lab products found in correlation
3 protocols using 1420 victor 2
1
Mitochondrial H2O2 Production Assay
2
Serum Spike Protein Antibody ELISA
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Twenty-five μL of serum as such or 25 μL of a solution of formulated powders at 250 mg/mL in cell culture medium (resulting in a 1:4 dilution with respect to protein concentration in the starting serum) were tested at several dilutions on ELISA plate wells coated with soluble full-length Spike protein, deleted of the transmembrane domain, and expressed in HEK293T cells as previously described [22] . Antibodies detected were expressed as the optical density at 450 nm measured by a Wallac 1420 Victor 2 microplate reader (Perkin Elmer, Milan, Italy).
3
Cartilage Explant Viability Assay
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Tissue culture media for explant wells A, A+, C, C+, CA, and CA+ was analyzed from 8 animals for PGE2 and c3a/c3a des Arg. Tissue culture media from C, C+, CA, and CA+ wells from all 16 animals was also analyzed for glycosaminoglycan (GAG) and nitric oxide (NO). Cartilage explants were stained with calcein-AM and ethidium homodimer-1 for an estimate of cell viability from all 16 animals.
All spectrophotometry and fluorescence readings were obtained from a microplate reader (1420 Victor 2, PerkinElmer; Fusion α, PerkinElmer). All chemical reagents were purchased from Sigma-Aldrich (Mississauga, ON, Canada) unless otherwise stated.
All spectrophotometry and fluorescence readings were obtained from a microplate reader (1420 Victor 2, PerkinElmer; Fusion α, PerkinElmer). All chemical reagents were purchased from Sigma-Aldrich (Mississauga, ON, Canada) unless otherwise stated.
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