Glowmax luminometer

Luciferase assays were performed following the protocol described previously [45 (link)]. Briefly, HeLa-PKR^kd cells (5 × 10⁴ cells/well) were seeded in 24-well plates a day before the transfection. For each transfection reaction mix, 0.05 µg of firefly luciferase (Promega) and 0.2 µg of each of the indicated PKR plasmids were co-transfected with 0.8 µg of either E3L, CRV157, CRV157-K48A/K49A, CRV157 del C, or CRV157 del N plasmids using GenJet (SignaGen Laboratories, Frederick, MD), with transfection reagent to DNA ratios of 2:1. For the titration experiments, the indicated concentrations of CRV157 or CRV157 del C plasmids were co-transfected. Each transfection was performed in triplicate, and plasmid DNA concentrations for each transfection mix were kept constant by adding an appropriate amount of pSG5 vector plasmid where necessary. After 48 h, whole-cell lysates were extracted using mammalian lysis buffer (GoldBio, St Louis, MO), USA, followed by the addition of luciferin (Promega, Madison, WI, USA). Luciferase activity was determined in a Glowmax luminometer (Promega). Each experiment was conducted at least three times, and representative experiments are shown.

On the day of treatment, spheroids were incubated with NB-PS conjugates (25, 50 and 100 nM in duplicates) in DMEM without phenol red (+10% FBS and Pen-Strep) for 2 h at 37 °C in the dark. Immediately after removing unbound conjugate, spheroids were irradiated at room temperature with a total light dose of 20 J/cm² (±690 nm, fluence rate of 7 mW/cm² confirmed by an Orion PD Optometer, total illumination time of 48 min). Subsequently, two untreated wells were exposed to 1% TritonX-100, serving as a control for 0% viability, and spheroids were incubated for 24 h at 37 °C. Bright field images of spheroids 24 h post NB-targeted PDT were taken on an EVOSfl or Nikon Eclipse digital inverted microscope using a 20× objective.
The next day, spheroid-containing microtiter plates were incubated with CellTiter-Glo 3D reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Thereafter, the contents of the microtiter plate were transferred to a white 96-well plate (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) and the luminescence signal was measured using a GlowMax Luminometer (Promega).
Experiments were conducted in biological duplicates.

Human neuroblastoma (SK-N-SH) cells cultured in minimal essential medium (MEM) supplemented with 1% fetal bovine serum (FBS) and 1% penicillin (100U/ml)/streptomycin (100μg/ml) were differentiated with 10 μM all-trans retinoic acid for 7 days [32 (link), 44 (link)]. After resting for 24h in the low serum medium, the cells were seeded in 24 well (10⁵cells/well) culture plates in fresh medium and incubated with 50μM or 500μM of individual GA or control peptide in non-covalent mixture with Pep-1 or Pep-1 alone for additional 24h. The cultures were then photographed using a phase contrast Leica microscope (Leica Microsystems Inc, Buffalogrove, IL). Subsequently, the cells were harvested, lysed with lysis buffer (M-PER, Pierce) and the lysate was assessed for metabolic activity using the luciferase based CTG assay (CTG, Promega, Madison, WI). Briefly, cell lysate (5μL) in 25μL of phosphate buffered saline was transferred to an opaque white 96-well plate, and then 30μL of CTG assay solution was added. The relative luminescent signal (RLT) was quantified using a Glowmax luminometer (Promega).