Spectramax id3

ROS/RNS analysis was performed using an OxiSelect In Vitro ROS/RNS Assay (Cell Biolabs, San Diego, California, USA) as per manufacturer’s instructions. The mean fluorescent intensity at 480 nm (excitation) and 530 nm (emission) were measured using a fluorescence plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA).
A secondary method of hydrogen peroxide analysis was performed using an Amplex Red Hydrogen Peroxide/Peroxidase Assay (ThermoFisher Scientific, Waltham, Massachusetts, USA) as per manufacturer’s instructions. The mean fluorescent intensity at 530 nm (excitation) and 590 (emission) were measured using a fluorescence plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA).

Biofilm formation was analyzed according to O’Toole (2011) . Overnight grown cultures were washed in 1xPBS and OD₆₀₀ was adjusted to 0.1. Bacterial strains were cultivated in a microtiter plate in 100 μl modified M9 minimal medium with 0.5% casamino acids (Biozol Diagnostica Vertrieb GmbH, Germany) without shaking at 28°C. After incubation OD₆₀₀ was measured in the plate reader (SpectraMax iD3, Molecular Devices). After 24 h OD₆₀₀ was measured and unattached cells were dumped out of the plate. The plate was washed twice by submerging it in MilliQ water to further remove unattached cells. Hundred and twenty-five microliter of 0.1% crystal violet (Roth, Germany) solution was added to each well. After 15 min the plate was rinsed three times in MilliQ water and dried for 1.5 h before visual inspection of biofilm production. For quantification of the biofilm 125 μl of 30% acetic acid (Roth, Germany) was added to each well and incubated for 15 min at room temperature. The solution was transferred to a new microtiter plate and color intensity was quantified at the plate reader (SpectraMax iD3, Molecular Devices) with absorbance at 550 nm and 30% acetic acid as blank. Biofilm-forming Pseudomonas simiae WCS417 served as positive control and non-biofilm-producing Escherichia coli DH5α served as negative control. The experiment was repeated three times with 6–12 replicates per strain.

For analysis of protein degradation of IKZF1, CK1α, SALL4 and PLZF, HEK293T cells stably expressing IKZF1-HiBiT, CK1α-HiBiT, SALL4-HiBiT, or PLZF-HiBiT were cultured in 96-well plates and treated with DMSO or thalidomide derivatives for 24 h. Then, the cells were lysed using Nano-Glo HiBiT Lytic Detection System (N3040, Promega) according to the manufacturer’s instruction. The luminescence signals of HiBiT-tagged neosubstrates were detected using SpectraMax iD3 (Molecular Devices).
For analysis of protein degradation of BRD3 cells stably expressing BRD3-HiBiT, they were cultured in 96-well plates and treated with DMSO or PROTACs for 6 h. Then, the cells were lysed using Nano-Glo HiBiT Lytic Detection System according to the manufacturer’s instruction. The luminescence signal of the HiBiT-tagged neosubstrate was detected using SpectraMax iD3 (Molecular Devices).

MCF-7 and T47D cells were seeded at 1 × 10³ cells and treated with 10⁻⁹ to 10⁻⁶ mol/L X15695 for 48 hours. Afterward, cells were loaded with 10 μmol/L H2DCF-DA in phenol red-free medium for 40 minutes. Cells were subsequently washed with medium and further incubated in medium for 120 minutes before analysis of fluorescence at 485 nm excitation and 530 nm emission using a multi-well fluorescence reader (SpectraMax iD3, Molecular Devices with SoftMax Pro 7 software). After the DCF assay, cells were frozen at −80°C overnight. After thawing, DNA was stained with a Hoechst dye 33258 (10 μg/mL final concentration) for 30 minutes at room temperature and fluorescence was measured at 352 nm excitation and 454 nm emission in a multi-well fluorescence reader (SpectraMax iD3, Molecular Devices with SoftMax Pro 7 software). DCF fluorescent intensities were normalized to Hoechst fluorescent intensities.

EVs were obtained from parent or DPH2KD HeLa cells transfected with a plasmid encoding NLuc-Hsp70. Acceptor parent HeLa cells were seeded in 96 well plates the day before EV incubation. The same protein amount of EVs for each condition is incubated with acceptor cells for 24 h. After incubation, acceptor cells are washed twice in DPBS and NanoLuc activity was measured in each well using the Nano-Glo Live Cell Assay System (Promega, Wisconsin, USA) following the manufacturer’s instructions using the iD3 SpectraMax microplate reader (Molecular Devices, California, USA). EV uptake is shown as percent of input material. Statistical significance is obtained through an unpaired t-test.

Mouse subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from whole hearts by differential centrifugation at 24 h post-LPS, as previously described [35 (link), 36 (link)]. Mitochondrial respiration was assessed using a Clark-type electrode (Hansatech, UK) at room temperature after the addition of the substrates of complex I (malate and glutamate) or complex II (succinate and rotenone) to assess basal respiration (state 2). Maximal oxygen consumption (state 3) was activated by the addition of 200 µmol/L ADP. Oxygen consumption was expressed as nmols O2/min*CS. Hydrogen peroxide (H₂O₂) production was assessed from changes in fluorescence after the addition of respiration substrates in the presence of Amplex® Red (10 µmol/L) and horseradish peroxidase (5 U/mL). Changes in fluorescence over time were monitored at 590 nm using a multimode reader (iD3 SpectraMax, Molecular devices). H₂O₂ concentration was calculated using standard H₂O₂ curves and rates were expressed as pmol H₂O₂/min/mg protein.