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After 24 hours, whole-cell lysates prepared with passive lysis buffer were collected for reporter detection by the Dual Luciferase Reporter System (Promega) according to the manufacturer's protocol.
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Then, the buffer was collected for NO measurement using the DAN method.
The wash buffer was collected for protein quantification using indirect ELISA.
An EDTA sample with 'magic buffer' was collected for genomic analysis (7D only).
100 mM NaCl/50 mM Tris HCl buffer was collected following the same procedures to obtain fraction NaCl-2. 1 M NaCl/50 mM Tris HCl buffer was collected for the last elution to obtain fraction NaCl-3.
The cells were washed and incubated with control or NMDA in the above buffer for 1 h, after which, the buffer was collected for NO measurement using the DAN method.
The buffer was replaced with a fresh buffer containing 3.7 mM glucose for one hour before the buffer were collected (basal) and replaced with a buffer containing 16.7 mM glucose for an additional one hour (glucose stimulated).
The proteins were separated from the beads using IB loading buffer, then the supernatants were collected for IB.
Red cells were removed by washing the cells in red cell lysate buffer for 4 times and the remains were collected for RNA extraction (Tanha et al. 2002; Miyazaki et al. 2015).
The protein was dialyzed extensively against the same buffer used for NMR, and the background diffraction from the buffer was collected and subtracted.
Protein samples were collected for Western blotting analysis in lysis buffer.
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