After stimulation for 24 h, the media from cells stimulated by LPS and IFN-γ with or without magnesium (5 mM) treatment were collected and centrifuged at 1500 rpm, and the obtained supernatants were stored at −80 °C for use in subsequent experiments with conditioned media.
The yellow/brownish suspension of silver nanoparticles was then filter purified to remove excess PHMB and other impurities using centrifuge filters (Amicon® Ultra centrifugal filters by Millipore Corporation, Billerica, MA, USA ) having a molecular weight cutoff value of 30 KDa and then stored at room temperature for further analysis and use in subsequent experiments.
The aerial parts were excised using a sterilized knife, and the residual root portions were transferred to sterilized plastic bags (12 × 24 cm), which were labeled, sealed, placed in an icebox, immediately transported to the laboratory, and stored at 4 °C until use in subsequent experiments.
Fractions containing the rM antigen were pooled for use in subsequent experiments.
For each PARP mutant, a clone closely matched to the PARP-WT cell line for both hPARP-1 by western blot (Figures 3B [left panel], 4A [left panel], and 5A [left panel]) and TRPM2 by whole cell current amplitude (Figure 3B [right panel], 4A [right panel], and 5A [right panel]), was selected for use in subsequent experiments.
EGFP+ cells were sorted by flow cytometry and cultured for use in subsequent experiments.
Resulting methanolic extracts were filtered, concentrated, dried, and stored in a desiccator for use in subsequent experiment.
Third-passage cultures (P3) were used in subsequent experiments.
The test-shock used in subsequent experiments was chosen to elicit 50% of the maximal slope.
From this control experiment, we can determine the suitably diluted UrD solution to be used in subsequent experiments.
Then, biological assays were conducted to determine a non-lethal dose used in subsequent experiments.
