Nano glo

The CD271 promoter region was amplified from the genome of HPCM2 cells using polymerase chain reaction and cloned into the pNL1.1-Nluc-Neo vector (Promega). 293 T cells were transfected with the plasmids using FugeneHD (Promega) according to manufacturer instructions, and 2 days after transfection, cells were lysed with Nanoglo (Promega), and luminescence was measured using a Synergy H1 system (Agilent Technologies). The pGL4.10[luc2] vector (Promega) was also transfected along with pNL1.1-Nluc-Neo for normalization.

Emission spectral scans of the HEK-293T cells expressing recombinant Nluc fusions were performed in 384-well F bottom Clear plates using a SpectraMax M5 fluorescence microplate reader (Molecular Devices, CA), with Dulbecco’s modified Eagle’s medium without phenol red, supplemented with furimazine and the assay substrate Nano-Glo at 100-fold dilution (Promega). Emission spectra were recorded from 380 to 650 nm using an integration time of 500 ms with 5-nm step increments. All spectra were normalized to the luminescence value at the emission maximum (449 nm) of Nluc.

Transiently transfected HEK293T cells were washed and maintained in complete HBSS media (ThermoFisher) supplemented with 0.035% Sodium Bicarbonate (ThermoFisher) and 0.38% HEPES. HEK293T cells were then co-incubated for 2 hours with RBM-derived peptides (0.5, 5 or 50 μM, final concentrations) and 0.1 μM (final concentration) His-tagged SARS-CoV-2 Spike protein RBD (GenScript) or vehicle. Following 2-hour incubation period, HEK293T cells were further incubated for 10 minutes at 37°C with the nLuc substrate Nanoglo (Promega) prior to measurement of total luminescence with the BMG Clariostar lab tech plate reader. The internalisation of membrane bound nLuc-ACE2, induced by S_RBD protein, is indirectly detected by a reduction in luminescence signal.

pGL4.20, pNL1.1 and pNL1.2-transfected cells were lysed after stimulation using Cell Culture Lysis Reagent (CCLR; Promega). Nano-Glo (Promega) was used to measure luminescence of the pNL1.1 and pNL1.2 transfected cells. Luminescence of pGL4.20 transfected cells was determined after addition of Luciferase Assay Reagent (Promega). Nano-Glo and Luciferase Assay Reagent were added to 30 μL lysate at 1:1 and 1:2 ratio, respectively. Medium samples were taken from cells expressing the pGLuc-basic plasmid. The amount of luminescence was assessed after the addition of BioLux GLuc Substrate (NEB) in a 1:1 ratio to 30 µL medium sample. All luminescent measurements were performed with the Tristar² LB942 (Berthold Technologies, Bad Wildbad, Germany). pDD-tdTomato expressing-cells were treated with 1 μM shield1 (kindly provided by Dr. Bongers, Radboud University) at the start of stimulation to stabilize the newly produced DD-tdTomato protein. Fluorescent measurements were carried out with a CLARIOstar monochromator (BMG Labtech, Ortenberg, Germany) by exciting the cells at 550 nm and measuring emission at 590 nm. 20 μL of total cell lysate was used for β-Gal colorimetric quantification to correct for differences in transfection efficiency (Invitrogen). Absorbance was determined using a Multiskan FC Microplate Photometer (ThermoFisher Scientific) at 405 nm.

SH-SY5Y cells were seeded on 6-well culture plates at 3 × 10⁵ cells per well. 24 h post seeding, each well was transfected using 7.5 μL FUGENE HD (Promega) and 1.25 μg nanoluciferase reporter plasmid along with 1.25 μg pGL4.13 (internal transfection control that encodes firefly luciferase [FFluc]) in 300 μL of OptiMEM (Invitrogen). Transfections of differentiated cells were performed on day 5 in RA supplemented media. Cultures were allowed to grow for 24 h after transfection. Cells were lysed in 250 μL Glo Lysis Buffer (Promega) for 5 min at room temperature. 50 μL lysate was mixed with 50 μL prepared Nano-GLO or ONE-Glo (Promega) for 2 min, and bioluminescence was detected using a GloMax® 96 Microplate Luminometer. Nanoluciferase signal was normalized to FFluc signal in each sample. pcDNA 3.1 encoding nLuc the AUG start codon mutated to a GGG (GGG-nanoLuc) was run in parallel with each experimental nLuc plasmid and subjected to both conditions to serve as a control for normalization.