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DNA was extracted from a centrifuged pellet by mechanic lysis using glass beads, followed by Nuclei Lysis Solution and Protein Lysis Solution (Promega) and 10% PVP solution to eliminate phenols.
Blood was then incubated in red blood cell lysis solution (Biolegend) for 10 min.
Briefly, 1×106 cells washed in ice-cold PBS were resuspended in 100 μl of HK cell lysis solution.
Noradrenergic nuclei from the brainstem were homogenized in lysis solution (Sigma Aldrich, St . Louis MO) and protein concentrations were determined using a micro BCA assay (Pierce, Rockford, IL).
The cell pellets containing leukocytes were re-suspended in 1 mL red blood cell lysis solution (BD) on ice for 2 min.
e The cells in microwells are lysed by inputting cell lysis solution through the microfluidic channel.
The lysis solution provided by the manufacturer contained benzalkonium chloride and a proprietary ATP-releasing agent.
Fifty microlitres lysis solution (0.5% TritonX-100 only) was used as blank reference.
After this incubation period, 50 μL of MTT lysis solution (DMSO) was added into each well.
Then the slide was immersed in cold lysis solution to lyse the cells.
After washing, the red blood cells were ruptured with 2 mL lysis solution (FACS lysing solution, BD Bioscience).
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