Multiplate reader

Telomerase activity was assessed by using the TeloTAGG Telomerase PCR ELISA Kit from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) as described before [21 (link)]. Briefly, isolated CD4+/CD8+ T cells were lysed in lysis buffer (10,000 cells/µL) for 30 min, at 4 °C, and then centrifuged at 16,000× g, 20 min at 4 °C. The protein concentration was determined in the supernatant according to the method of Bradford [22 (link)]. For the PCR reaction, a total volume of 50 µL with equal amounts of protein was used and the products were amplified using 30 cycles (94 °C for 30 s, 50 °C for 30 s and 72 °C for 90 s). Each sample was run in duplicate; as negative controls, heat-treated samples, nuclease-free water, and lysis solution were used. Five microliters of the PCR product were used for the ELISA assay. Absorbance was measured at 450 nm with a reference wavelength of 690 nm using a multiplate reader from Tecan (Tecan Group Ltd, Crailsheim, Germany).

Each treated membrane was placed in a defined Trastuzumab solution of varying concentrations to estimate the q_max, via a Langmuir isotherm fitting. Trastuzumab concentrations of 0.5, 0.3, 0.2, 0.1, 0.05, 0.025, 0.01, and 0 mg mL⁻¹ in PBS pH 7.4 were prepared. The membranes were cut to a 6 mm diameter, placed in 0.25 mL of each concentration, and incubated for 1 h at 25 °C and 1000 rpm. After this, a supernatant analysis and direct analysis of the membranes occurred via UV-vis and BCA assay, respectively. For the direct analysis of the ligand load on the membranes via BCA, the membranes were incubated on a 96-well filter plate with the working reagent and 25 µL of PBS buffer. After incubation, the filter plate was placed on top of a 96-well plate and centrifuged. The resulting permeate was then analyzed with a multiplate reader from the Tecan Group (Männedorf, Switzerland) at 562 nm. The supernatant analysis was carried out via UV-vis at 280 nm.

The cell proliferation and viability to exogenous stimuli were spectrometrically quantified by using WST-1 reagent (Roche, Basel, Switzerland) according to the manufacturer’s instructions. A total cell number of 1700 per cavity of a 96-well culture plate (Greiner bio-one, Frickenhausen, Germany) were used and maintained in fully supplemented growth medium for 24 h before treatment with LPS and ATP. After 20 h, the WST-1 reagent was added to each well. The absorbance at the wavelength 440 nm and 590 nm as a reference were determined at time points 0, 1, 2, 3, and 4 h after WST-1 supplementation using a multiplate reader (Tecan, Männedorf, Switzerland).

Cell viability was measured using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) as previously described [17 (link)]. Briefly, 3 × 10³ cells (2 × 10³ cells for HCC827TRB10) were plated into each well of 96-well flat-bottomed plates and grown in RPMI-1640 containing 10% FBS. After 24 hours, dimethyl sulfoxide (DMSO), CDDP, GEM, DOC, PAC, VNR, and erlotinib with or without entinostat were added at the indicated drug concentration, and cells were incubated for an additional 72 hours. A colorimetric assay was performed after addition of 10 μl Cell Counting Kit-8 reagent to each well, and the plates were incubated at 37°C for 2–4 hours. Absorbance at 450nm was read using a multiplate reader (Tecan, Männedorf, Switzerland). Percent growth was expressed relative to DMSO-treated controls.

After GSH measurement, the DNA content of the cells was quantified using the blue fluorescing nucleic acid dye Hoechst 33,258 (FluoReporter™ Blue Fluorometric dsDNA Quantitation Kit (F2962), Invitrogen™, Thermo Fisher Scientific, MA, USA). For cell lysis, the medium was replaced by 100 µL dH₂O before the samples were frozen at − 20 °C. Later, they were incubated for one hour at 37 °C before 100 µL of the staining solution (prepared as described by the manufacturer) were added immediately before measuring the fluorescence at Ex/Em 360/460 nm using a multiplate reader (Tecan). All results were normalised to the first day values of the control group cultured in darkness.

Reduced levels of intracellular glutathione (GSH) can be interpreted as an indirect measurement of ROS concentration. GSH levels were measured using the optimised monochlorbimane (MCB) assay as previously described^{32 (link),33 (link)}. On day 1, 3, and 7 of culture, 240 µM MCB (Sigma-Aldrich, Germany) in PBS were added directly to the medium to achieve a final concentration of 40 µM and fluorescence was recorded immediately at Ex/Em 394/490 nm over 12 min using a multiplate reader (Tecan). All results were normalised to those of the control group cultured in darkness on day 1. Furthermore, GSH levels were related to the DNA content of the respective sample.