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After reviewing the literature, we suggest sampling at least every 15 min following an initial waking sample, for at least 1 h after waking.
The temperature stability of the experiment was ensured by storing the sample for at least 2 weeks at room temperature prior to scanning and by cooling the imaging sample with a water cooling device.
We then added 2 mL of the freshly made staining solution, containing per sample, 2 mL staining buffer, 12 μL Propidium Iodide, and 6 μL of RNAse A stock solution (5 mg Rnase combined with 1.5 mL water) (all solutions included in the kit), and incubated the sample for at least 30 min, shielded from light.
A sample was considered to have failed QC and removed from analysis (n = 8 cases and 2 controls) if it had an extremely large number of CNV calls, defined as having a value greater than three standard deviations from the mean of the log-transformed number of CNV calls per sample for at least one of the algorithms.
The fluorescence signal produced by free AMC is proportional to the caspase-3 activity present in the sample, for at least the first 10 h, and this was monitored using a fluorescence plate-reader (FL800, Bio-Tek Instruments INC, Germaty) at 360/40 nm excitation and 460/20 nm emission wavelengths.
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Centrifuge the sample for 20sec at full speed, at 4°C.
Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2 h.
However, these subjects were included in the analysis as they had adequate samples for at least one time point.
Inclusion criteria were length of stay in the ICU ≥ 72 hours and complete data available on serum and urinary samples for at least 3 days.
Inclusion criteria were age ≥18; non-traumatic arrest; complete data available on serum and urinary samples for at least 3 days; survival ≥ 72 hours after admission.
Inclusion criteria were as follows: age ≥18; non-traumatic arrest; complete data available on serum and urinary samples for at least 3 days; survival ≥72 h after admission.
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