Luminometer centro lb960

U87 cells were cultivated in DMEM/10% FBS, while cells from grade 4 GBM tumors were cultivated in 50% hCSF, 45% DMEM, and 5% FBS in 96-well plates (Cell+™, Sarstedt, Nümbrecht, Germany). Infection of cells with pseudotypes was carried out as described previously [29 (link)]. Briefly, cell culture supernatants from COS-1-transfected cells were given to tumor cells at a 70–80% density. After 3 h, 100 µL of medium was added, and the cultures were incubated at 37 °C for 3 days. For luciferase detection, the cells were washed three times on day three, and 100 µL of the BrightGlow™ luciferase assay reagent (Luciferase Assay System, Promega E1483, Walldorf, Germany) was added. The plates were incubated for 5 min, and bioluminescence was measured using a 96-well plate reader (Luminometer Centro LB960, Berthold Technologies, Bad Wildbad, Germany). The green fluorescence of HIVgfp-infected cells was monitored using an EVOS M7000 Imaging System (Thermo Fisher Scientific, Braunschweig, Germany).

Arabidopsis protoplast transformation and luciferase assay was performed as previously described^{82 (link)}. Promoter fragments were cloned into pSK-vectors with C-terminal luciferase. GUS expression under 35S-promoter was used as a transformation control. GUS and LUC measurements were performed using a microplate reader (luminometer Centro LB960; Berthold Technologies, Bad Wildbad, Germany, www.berthold.com). Data were calculated as ratios of LUC to GUS.

For this analysis, AFSC were seeded at a density of 3000 cells/well in 300 µL DMEM, supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin/neomycin) in plates with 96 wells. After 24 h of seeding, the cells were treated with the synthesized Ag/Fe-enhanced TiO₂ powders and incubated for 72 h. The GSH-Glo Glutathione Assay kit (Promega, WI, USA) was used for oxidative stress assessment. The kit measures the amount of glutathione (GSH) that is produced by cells and converted to oxidized glutathione. The amount of glutathione transformed is directly proportional with the amount of glutathione S-transferase (GST) enzyme that catalyzes the oxidation reaction, which is also linked to the formation of a luminescent luciferin. If the light emission is poor, glutathione production is inhibited and thus oxidative stress is increased, while in the case of an intense light, more glutathione has been synthesized and thus the cell is less stressed.
Initially, 100 µL 1X GSH-Glo Reagent was added, followed by incubation at 37 °C for 30 min. Subsequently, 100 μL Luciferin Detection Reagent was added and incubated at 37 °C for another 15 min. After 15 min, the medium was well homogenized and the plate was read on the luminometer (Microplate Luminometer Centro LB 960, Berthold, Germany) [31 (link)].

The cells were cultured in a white-wall 96-well Costar plates (Corning, Somerville, MA, USA) as required cell density in order to triplicate each condition (Non-Treated, Light Only, 5-ALA Only, PDT Treated). 100 µL of Celltiter-Glo mix (Promega, Madison, WI, USA), which determines the cellular viability in a culture based on the quantification of the ATP present, was prepared according to the manufacturer’s instructions, was added to each well and incubated at room temperature for 10 min in the dark. Luminescence reading was performed using the Luminometer centro LB960 (Berthold Technologies, Oak Ridge, TN, USA) running MikroWin software Version 4.41 (Mikrotek Laborsysteme GmbH, Overath, Germany). Results were expressed in relative luminescence units (RLU) or normalized RLU. Normalized RLU = RLU of the sample/Average RLU of Non-Treated control.

In brief, HEK 293T cells were seeded at a density of 1 × 10⁵ cells in a 24 well plate and incubated for 24 h. Thereafter, cells were co-transfected with pGL3b-kB4, a luciferase reporter plasmid and pRL-TK, a thymidine kinase promoter-Renilla luciferase reporter plasmid using lipofectamine LTX plus transfectiont reagent (Invitrogen, Life Technologies, USA) according to manufacturer’s instructions. Cells were subjected to treatments as described in the figure legends. The NF-κB luciferase reporter assay was performed with Luciferase Assay Kit (Promega, USA) as per manufacturer’s instructions. At the end of incubation, cells were lysed in 100 μl of lysis buffer and cell lysate was mixed with 1:5 ratio of luciferase assay reagent (LARII). The values of firefly and renilla were recorded in a Luminometer (Centro LB 960, Berthold, USA). The results are expressed as the ratio of firefly luciferase activity to that of renilla and normalized to protein concentration.

In antiproliferative assays, compounds were assayed for their growth inhibiting activity towards the described cancer cell lines using the CellTiter-Glo Luminesent Cell Viability Assay as described by the manufacturer (Promega Corporation). Briefly, 5 × 10³ cells were plated onto 96-well plates (white with clear bottom (3610, Corning Costar) with 90 μl media per well and incubated overnight. Compounds were added at different concentrations (varying from 100 to 0.032 μM) to each well and cell cultures were incubated at 37°C during 72 h. Vehicle (DMSO) was used as control and all compounds were tested in constant percentage of DMSO (0.5%). After addition of 50 μl CellTiter GLO, luminescence was measured using a Centro luminometer LB960 (Berthold). Dose–response curves were generated and 50% inhibitory concentration values (IC50) were calculated using nonlinear regression analysis (Graph Pad Prism).

Caspase 3/7 activation was analyzed in HeLa cells treated with the indicated compounds for 16 h using the Promega Caspase Glo assay. Briefly, 5 × 10³ cells were plated onto 96-well plates in 100 μl of media per well 6 h before the assay. Compounds were added at 50 μM to each well, and cell cultures were incubated at 37°C for 16 h. Vehicle (DMSO) was used as a control, and all compounds were tested at a constant percentage of DMSO (0.5%). After adding 50 μl of Caspase 3/7 GLO, luminescence was measured using a Centro luminometer LB960 (Berthold).