Glomax detection system

A NanoLuc MICA shedding assay was performed as reported previously with slight modifications^{23 (link)}. Briefly, cells seeded onto 10 cm dishes were transfected with 2.0 µg N-terminal NanoLuc-tagged MICA-expressing plasmid, incubated for 12 h, reseeded onto a 96-well plate, and incubated for another 12 h. After application of URB597 or DMSO as the control, the culture medium was collected and mixed with the luminescent substrate (Nano-Glo Luciferase Assay System; Promega), and luminescence values (Luc_sup) were measured using the GloMax Detection System (Promega). The cells were washed with PBS, and the luminescent substrate (Promega) was added to determine the luminescence values (Luc_cell) using the GloMax Detection System. Soluble MICA/B levels were determined as Luc_sup/(Luc_sup + Luc_cell) to adjust for transfection efficiency.

7 × 10⁴ HEK293T cells were seeded in 24-well plates, KD was induced immediately and cells were transfected with 500 ng of firefly luciferase reporter gene plasmid and 100 ng of renilla luciferase plasmid. After 72 h cells were harvested and prepared for measurement using the Dual Glo luciferase assay kit (Promega) as recommended by the manufacturer. Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values to exclude variations in transfection efficiency. For testing the secretion efficiency, cells were transfected with 500 ng gaussia luciferase and 100 ng of renilla luciferase plasmid. Luciferase activity was measured in cell culture media and normalized to intracellular renilla luciferase activity.

ADV-GFP was amplified in PK-15 cells, while HSV-GFP and VACV-GFP were amplified in Vero cells and quantified using a plaque assay. The Sendai virus Cantell strain, GFP-tagged H₁N₁ virus-PR8 strain (PR8-GFP), and NDV-GFP were amplified in specific pathogen-free (SPF) eggs. Before virus infection, the culture medium was replaced with DMEM containing 1% FBS, and the virus was introduced into the target cells at a specific multiplicity of infection (MOI). After a 2 h incubation at 37 °C, the extracellular viruses were eliminated, and the medium was substituted with DMEM containing 10% FBS. At the specified time points, the cells were detached from the culture plates along with the supernatants, and the mixture was subjected to centrifugation at 3000 rpm for 3 min. The supernatant of each sample was separated from the cell pellet for ELISA. The cell pellet was reconstituted in 300 µL of phosphate-buffered saline (PBS), and the fluorescence of each sample was assessed using a fluorometer, specifically, the Glomax detection system from Promega. The plasmids were introduced into PK-15, PAMs, PIBs, and MA104 cells using Lipofectamine 2000 (Invitrogen), and for HEK293T and 293-Dual™ hSTING-A162 cells, Polyethylenimine (PEI; Polyscience Inc., Warrington, PA, USA, 23966) was employed for transfection. The procedures followed the manufacturer’s protocol.