Plate reader luminometer

Attached and detached retinas were harvested after 3 days and homogenized using a sonicator at 20% power for 10 pulses in lysis buffer (20 mM MOPS, pH 7.0; 2 mM EGTA; 5 mM EDTA; 0.1% Triton X-100). 1 tablet of protease inhibitor (Complete Mini; Roche Diagnostics, Indianapolis, IN) and 1 tablet of phosphatase inhibitor (PhosSTOP; Roche Diagnostics, Indianapolis, IN) per 10 mL were added to the lysis buffer. The homogenates were centrifuged at 10,000 g for 10 minutes at 4 °C, and total protein concentration of the supernatant was determined using a Micro BCA protein assay kit (ThermoScientific, Rockford, IL). A luminescent assay kit (Caspase 8 Glo Assay, Promega, Madison, WI) was utilized to measure caspase 8 activity according to the manufacturer’s instructions. In short, 50 μg of protein was incubated with substrate in a white-walled 96 well-plate at room temperature for 1 hour. The attached retinas served as controls. Luminescence was measured in a plate reader luminometer (Turner Biosystems, Sunnyvale, CA).

Two retinas for each mouse were pooled for detection of caspase 8 activity. After homogenization and centrifugation at 10,000× g for 10 min at 4 °C, the protein concentration was quantified by the Dc Protein Assay kit as mentioned above. A luminescent Caspase-Glo^® 8 Assay kit (G8201, Promega, Madison, WI, USA) and a plate reader luminometer (Turner Biosystems, Sunnyvale, CA, USA) were used to measure caspase 8 activity.

Cells were seeded at 2000 cells per well in a 96-well U-bottom plate (#353077, Corning). The 3D cell culture media was a mixture of the media for 2D cell culture supplemented with 10% Matrix (#A1413201, Thermo Scientific). On day 6, sphere colonies were firstly observed under microscope and images were taken using Leica microscope with X 5 bright field objective. The length and width for each spheroid were measured afterwards in Image J (v1.53k). The viability for spheroids was determined by CellTiter-Glo^® 3D Cell Viability Assay Kit (#G9681, Promega). Briefly, 100 μl CellTiter-Glo^® 3D Reagent was added into the wells to be determined, followed by shaking for 5 min. After incubation at room temperature for 25 min, the plate was read on the luminometer plate reader (Promega). The luminescence signals were measured and collected for evaluating the 3D cell growth viability.

Purified influenza virion was diluted to 5 μg/mL and was used to coat the bottom of a Maxisorp 96-well ELISA plate (ThermoFisher). Virus was allowed to bind to the plate overnight at 4°C, and then the plate was blocked with blocking buffer (1% milk in PBS-Tween). Sera was diluted 1:100 in blocking buffer and then serially diluted 3-fold. Plates were incubated with sera for 2 h, after which the plates were washed 3 times with blocking buffer. Plates were incubated with goat α-mouse IgG-HRP secondary antibody (Prometheus) diluted 1:3,000 in blocking buffer and incubated for 1 h. Plates were washed 3 times with blocking buffer and then incubated with 100 μL of SigmaFast OPD substrate for 30 min. Substrate reaction was stopped with 25 μL of 3 M HCl, and absorbance was measured at 450 nm with a Luminometer plate reader (Promega).