Synergy neo2 reader

Hedgehog activity was analyzed in C3H10T1/2 or HEK293T cells by using a GLIBS reporter plasmid, expressing firefly luciferase in a manner dependent on the activity of Hedgehog pathway. Full growth medium was replaced 24 h after transfection and cells were lysed in lysis buffer (Promega) 48 h after transfection. Cell lysates were incubated with Stop & Glo substrates (Promega) and luciferase activity was measured in a Synergy Neo2 reader (Biotek).

pGL3-ICAM-1 luciferase reporter (pGL3-ICAM-1-WT) was a generous gift from Dr Jim Hu at the Hospital for Sick Children in Toronto, ON, Canada (Yu et al., 2015 (link)). QuikChange Lightning Site-Directed Mutagenesis (Aligent) was performed as described (Alam et al., 2020 (link)) to mutate the STAT3 binding site within the ICAM-1 promoter from 5′-TTC-CxG-GAA-3′ to 5′-AGC-CxC-CTG-3′. Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 (http://n2t.net/addgene:24983; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) (Hillion et al., 2008 (link)). HUVECs seeded at 1.5×10⁶ cells per well of a collagen-coated six-well plate were transfected with plasmids using the Neon Transfection System (Invitrogen), harvested 48 h post-transfection. Then, Dual-Luciferase Reporter Assays (Promega) were performed, and firefly and Renilla luciferase luminescence was measured using a Synergy Neo 2 Reader (BioTek).

Between 0.5 and 15 mg (1.5 to 45 nmol) of human fibrinogen containing Factor XIII (Fisher Scientific) was dissolved in 0.4 to 1.2 ml of 100-mM ${\mathrm{NaHCO}}_3/{\mathrm{Na}}_2{\mathrm{CO}}_3$ pH 7.5 to 9 buffer, and 0.005 to 0.2 mg (5 to 150 nmol) of IRDye 800CW NHS ester (Li-Cor Biosciences) was dissolved in dimethyl sulfoxide at a concentration of 0.6 to $1\mu \mathrm{g}/\mu \mathrm{l}$ . The dye solution was mixed into the fibrinogen solution, and the vial was shaken for 3 to 5 h at room temperature to form 800CW-conjugated fibrinogen [ $\mathrm{Fgn}(800\mathrm{CW}{)}_x$ ]. The reacted solution was loaded into 50 to 100 kD molecular-weight cutoff (MWCO) centrifuge filter vials (Amicon, EMD Millipore), then repeatedly filtered and replenished with phosphate-buffered saline (PBS) or saline until the theoretical fraction of retained free dye was $<0.01\%$ . The labeling ratio of dye moieties per fibrinogen molecule, $x$ , was estimated by measuring the absorbance of the purified conjugate solution at 290 and 774 nm using a Synergy Neo2 Reader (BioTek Instruments, Inc.) and solving for the individual concentrations of dye and fibrinogen using standard curves of absorbance for the respective pure solutions.¹¹ (link)

An adrenochrome reporter end-point assay adapted from Cedrone et al. was used to measure the relative amount of hydrolyzed epoxide (Cedrone et al., 2005 (link)). Absorbance at 490 ​nm was measured using a TECAN Infinite M1000 reader or a Biotek Synergy Neo2 reader. All reactions were carried out at 37 ​°C with 20 ​μM protein and 2 ​mM substrate in 100 ​mM NaCl, 20 ​mM HEPES, pH 7.4, and 2% DMSO. When testing the activity of Cfl2 against biological substrates, 80 ​μM protein was used. The xenobiotic epoxide reactions proceeded for 1 ​h in a 100 ​μL reaction, and the poly-unsaturated fatty acid epoxide reactions proceeded for 2 ​h in a 50 ​μL reaction. The reactions were quenched by adding 0.5 ​× ​the starting reaction volume of a solution of 90% acetonitrile containing sodium periodate at a 1:1 ​M ratio to the starting substrate and incubating for 30 ​min ​at room temperature. A molar excess of epinephrine-HCl in 0.5 ​× ​the starting reaction volume was finally added, and an aliquot of 100 ​μL (xenobiotic epoxide reactions) or 90 ​μL (fatty acid epoxide reactions) was then used to measure absorbance at 490 ​nm in a transparent 96-well flat-bottom plate. Replicates for each experiment were obtained by performing the hydrolysis reactions on different days using the same preparation of each enzyme and making new enzyme and substrate working stocks for each replicate.

Activity of 45 different cellular signaling pathways was measured following the manufacturer’s instructions (Cat. no. CCA-901L-12; Qiagen). Briefly, wild-type and Pkd1- or Pkd2-null NIH3T3 cells, or wild-type and Pkd2-null mIMCD3 cells were seeded for reverse transfection in a 96-well plate containing immobilized reporter plasmids. Every pair of wells contained plasmids expressing firefly luciferase in a manner dependent on activity of a specific cellular pathway. Every well also contained a plasmid that constitutively expressed Renilla luciferase, to account for differences in transfection efficiency. Reverse transfection was mediated by Attractene (Cat. no. 301005; Qiagen). Full growth medium was replaced 24 h after transfection, and cells were lysed in lysis buffer (Cat. no. E194A; Promega) 48 h after transfection. Cell lysates were incubated with Stop & Glo substrates (Dual-Luciferase Reporter Assay System, Cat. no. E1960; Promega), and luciferase activity was measured in a Synergy Neo2 Reader (Biotek). All firefly luciferase values were normalized to Renilla luciferase values, and then all mutant sample values were normalized to wild type.