ATP levels were measured using the Bioluminescence Assay Kit (Roche) according to the manufacturer’s instruction as previously described (Yu et al., 2016 (link)). Briefly, the mice were anesthetized, and the brains were quickly removed. The hippocampi were homogenized using a lysis buffer followed by centrifuging at 12,000 r/min for 10 min. The ATP levels in subsequent supernatants were measured. The Cytochrome C Oxidase (CcO) activity of mitochondrial fractions was measured as previously described (Fang et al., 2015 (link)).
Bioluminescence assay kit
Manufactured by Roche
The Bioluminescence assay kit is a laboratory tool used to measure the amount of ATP (adenosine triphosphate) present in a sample. ATP is a molecule essential for energy transfer in living cells, and its detection can provide insights into cellular metabolism and viability. The kit utilizes the bioluminescent reaction between luciferase enzyme and its substrate, luciferin, to generate light proportional to the ATP concentration in the sample.
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3 protocols using bioluminescence assay kit
1
Measuring Hippocampal ATP and CcO Activity
2
Platelet Activation and ATP Quantification
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Human and murine platelets were isolated and activated with CRP or thrombin as described above and fixed for 2 h at 4°C using 2% Paraformaldehyde. The platelets were centrifuged at 650×g and releasates were collected and stored at −70°C before use. ATP levels were quantified using the bioluminescence assay kit from Roche Diagnostics following the manufacturer's protocol.
3
Quantifying Cellular ATP in Optic Nerve
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ATP extraction was performed as previously described (Khan, 2003 (link)) and modified for optic nerve as described in previous publications (Baltan et al., 2010 (link), 2011 (link)). In brief, nerves were diced and placed in 75 µl of 10% perchloric acid (HClO4), sonicated, and centrifuged at 4,500 rpm for 10 min at 4°C. The supernatant was neutralized with 30 µl of 2.5 M potassium hydroxide (KOH) and centrifuged at 14,000 rpm for 10 min at 4°C. Total cellular ATP was measured using a bioluminescence assay kit (11 699 709 001; Roche). The absorbance was measured at 560 nm. The blank values were subtracted from the raw data, and ATP concentrations were calculated from a log–log plot of the standard curve and were normalized to protein concentration. The values are expressed as micromoles of ATP per milligram of protein. Protein content was quantified in one nerve, and ATP levels were measured in the other.
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