Clarity luminescence microplate reader

HEK293T cells were seeded into 24-well plates. After 24 h, cells were transfected with overexpressed (pHAGE-6tag-C7ORF41) or knockdown (pLenti-shRNA#1 or pLenti-shRNA#3) vectors with 100 ng of NF-κB promoter fluorescent reporter plasmid and 2.5 ng of internal reference plasmid, phRL-TK. An empty lentiviral vector was used as a control. After 48 h of transfection, cells were stimulated with TNF-α for 6 h. Cells were then lysed, dissolved in luciferase assay buffer II, and centrifuged at 9729 ×g. The supernatant was collected, and the absorbance was measured with the Clarity Luminescence Microplate Reader (BioTek Instruments). The reaction was conducted with a dual-luciferase reporter gene detection kit (Promega, Madison, WI, USA) using the dual-luciferase reporter assay system (Promega).

To examine the activation of IP-10 promoter by SARS-CoV SUD, mock and transfected cells expressing SUD-FL, SUD-NM, and SUD-MC were co-transfected with CXCL10 promoter-driven firefly luciferase reporter plasmids (IP-10GL3, tIP-10GL3, and IP-10mκB1) and an internal control Renilla luciferase reporter pRluc-C1, as described in our prior report [37 (link)]. After a 1-day incubation, the cells were harvested and then dissolved in the lysis buffer; the activity of firefly and Renilla luciferases in the lysate was detected using Dual-Luciferase^® Reporter (DLR™) Assay System (Promega, Waltham, MA, USA) in Clarity™ Luminescence Microplate Reader (BioTek Instruments, Winooski, VT, USA).

Caspase 3/7 activity was directly measured at 48 and 72 hours after treatment using Caspase-Glo 3/7 Activity kit (Promega) according to the manufacturer’s protocol. Relative light intensity was measured in each well using Clarity Luminescence Microplate Reader (BioTek).

ATP concentrations were assessed using the ATP Bioluminescent Assay Kit (Sigma-Aldrich). Briefly, Leydig cells were isolated from Mrp4 animals and cultured overnight at 37° at a density of 5 × 10⁵ cells per well in a 12-well plate. Cells were treated with 500 μM 6 MP for one hour, washed with 1× phosphate buffered saline (PBS), then incubated with ATP lysis buffer (100 mM potassium phosphate buffer (pH 7.8), 1% Triton X-100, 2 mM EDTA, and 1 mM dithiothreitol) for 30 minutes at 37°. 2,4-Dinitrophenol (DNP) was used as a positive control54 (link). Luminescence was measured using the Clarity Luminescence Microplate Reader (BioTek Instruments).

Caspase 3/7 activity was directly measured at 24, 48 and 72 hours after treatment using Caspase-Glo 3/7 Activity kit (Promega) as per the manufacturer’s protocol. Briefly, the cells (5 × 10⁵/ml) were seeded into 24-well plated and treated with vehicle (DMSO) alone or ruxolitinib at various concentrations for 48 and 72 hours, Caspase-Glo reagent was added and cells were incubated for 1 hour at room temperature in the dark. Relative light intensity was measured in each well using Clarity Luminescence microplate reader (BioTek, Vermont, USA).

Luciferase reporter plasmids of the E2F1 promoter region (−483 to +25 bp) were constructed as pGL2-E2F1 plasmids. The pGL2-control plasmids were used as negative controls. Cells were transfected with 500 ng/well of pGL2 plasmid and 100 ng/well of pRL-TK using Lipofectamine (Invitrogen). After 24 hours, cells were transfected with the ANKRD22-plasmid. After another 48 hours, cells were harvested and analyzed for luciferase activity using the Dual Luciferase Reporter Assay (Promega) in a Clarity Luminescence Microplate Reader (BioTek). The relative luciferase activity (firefly luciferase) was normalized to pRL-TK activity (Renilla luciferase). Results were expressed as a fold induction over that of empty pGL2 activity.