Gaussia luciferase glow assay kit

Primary human T cells were heated in a thermal cycler and transferred to culture plates for incubation at 37 °C. Unless otherwise noted, cellular supernatant was sampled for luciferase activity 24 h after conclusion of thermal treatment. Non-thermal treatments were conducted by incubating engineered cells at indicated concentrations of CoCl₂ (Sigma 232696-5 G) or CdCl₂ (Sigma 202908). When indicated, luminescence was compared to a ladder of recombinant Gaussia luciferase (NanoLight 321-500) quantified using a Gaussia Luciferase Glow Assay kit (ThermoFisher 16161) according to the manufacturer’s instructions. For viability and proliferation studies, primary human T cells were heated in the thermal cycler before assaying with an apoptosis detection kit (BD 556547) or CellTrace Violet (Fisher C34571). Viability was assessed 24 h after heating and gating strategies are depicted in Supplementary Fig. 4. For migration studies, wild-type cells were added to the top insert of a transwell plate (Sigma CLS3421), while CXCL12 (50 ng ml⁻¹, Peprotech 300-28 A) was added to the lower chamber. Cells in the lower chamber were counted using a hemocytometer at indicated times.

Plasmids were transfected with TurboFect transfection reagent (Thermo Scientific). siRNAs were transfected at 10 nM final concentration with MISSION^® siRNA Transfection Reagent (Sigma-Aldrich). shRNA and siRNA co-transfections were performed using JetPRIME^® reagents (Polyplus Transfection). SEAP and luciferase activities in culture supernatants were quantitated using a Ziva® Ultra SEAP Plus Detection Kit (Jaden BioScience) and a Gaussia Luciferase Glow Assay Kit (Thermo Scientific), respectively. Luminescence signals were measured by a VICTOR^{3 (link)} Multilabel plate reader (PerkinElmer).