Nano glo luciferase assay substrate

To monitor bacterial lysis during the bacterial peptide treatments, we transformed E. coli BL21 with reporter plasmids expressing codon-optimized eGFP and NanoLuc^® Luciferase (Promega, Madison, WI, USA) genes under the control of the constitutive Pupp promoter. To monitor bacterial lysis using fluorescent microscopy, BL21 (pACYC184-GFP) cells were incubated with 10 µM of the antibacterial peptides, and microscopy was performed at time points 15, 30, and 60 min and at 4 h. All fluorescence images were taken at 488 nm excitation. The emission filter was 525/50 nM for cytoplasmic GFP. Phase-contrast images were also collected at the same time points. To monitor bacterial lysis in solutions by protein leakage, BL21 (pACYC184 nLuc) cells were washed with PBS (pH 7.4) three times, diluted with PBS to 10⁵ cfu/ml, and then incubated with peptides for 15, 30, and 60 min. After incubation, the intact bacterial cells were precipitated and the luciferase activity of the supernatant was examined using the Nano-Glo^® Luciferase Assay Substrate (Promega). The linearity of the assay was assessed using activity measurements of serial dilutions of the completely lysed BL21 (pACYC184 nLuc) bacterial culture. An undiluted bacterial culture was taken as 100%. Data were corrected to the background luciferase activity obtained with the supernatant of untreated cells.

The Leica DMi8 microscopy and Andor iKon-M 934 CCD camera were used to take pictures of luminescence from planarian cells and 293T cells with a 20x air objective (Leica, 506521). The 200,000 planarian live cells were resuspended in 50 μL culture medium and transferred to a glass-bottom dish (NEST, 801002) for imaging. The luminescence was measured after directly adding 1 μL Nano-Glo luciferase assay substrate (Promega, N1110) to cells. The exposure time was set to 20 s for luminescence imaging, and 1 s for bright field imaging.

Transcripts (5 μg) of a Jc1 derivative expressing Nanoluciferase (JC1-NLuc) (Amako et al., 2015 ) were electroporated into Huh7 cells (see Supplementary). Compound was added as in Section 2.3. Cell viability was performed as in Section 2.5 following 4% paraformaldehyde fixation of cells. NanoLuc was measured using a BMG Labtech plate reader following addition of 50 μl PLB and addition of equal volume of NanoGlo Luciferase Assay Substrate (Promega).

For production of VLPs, SH-SY5Y cells into which siRNA had been introduced were transfected with pCXSN-WNC, pCMV-WNV-prME, and pCMV-WNrep-NLuc(sec)2A. The culture supernatants were collected at 48 hpt and inoculated into Vero cells. After 48 h, the cells were treated with Nano Glo luciferase assay substrate (Promega) for 3 min and luminescence was measured using GloMax (Promega).