Infinite m200 pro multimode reader

Plasma AOPPs were determined using spectrophotometric detection according to (Witko-Sarsat et al., 1996 (link)) with minor modifications. Two hundred μL chloramine T-solution (0–200 µM) or 200 μL of plasma diluted 1:5 with PBS were mixed with 10 µL 1.16 M potassium iodide and 20 µL acetic acid. The absorbance at 340 nm was measured immediately using an Infinite M200 PRO multimode reader (TECAN, Männedorf, Switzerland).

The activity of caspase 3 and 7 was detected using the Caspase-Glo 3/7 Assay (Promega, Mannheim, Germany). The cells were seeded in white walled 96-well plates at a density of 15,000 cells per well and allowed to attach. Twenty-four hours after plating cells were treated in triplicate with 0.5 µM staurosporin and DMSO as solvent control (0.5 %). Caspase-3 and -7 activities were determined after 12 h after staurosporin treatment according to the manufacturer’s protocol. Caspase-Glo 3/7 reagent was added (25 µl/well), gently mixed, and incubated for 30 min. The assay is based on the cleavage of a proluminogenic substrate, containing a DEVD sequence, by the caspases 3 and 7 which generates a luminescent signal produced by luciferase. Luminescence, that is proportional to the amount of caspase activity present, was measured using an Infinite M200 PRO multimode reader (Tecan, Männedorf, Switzerland).

p-MIR reporter plasmid containing 3′UTR of KLF4 and plasmid containing the mutated sequence of KLF4 were purchased from Addgene. Dual-luciferase assay was performed using the DLR assay kit (Cat # E1910, Promega, Madison, WI, USA) as per the manufacturer’s protocol using plasmid expressing the Renilla luciferase gene (pRL, Promega, Madison, USA, 20 ng) as a control. Luciferase activity was assayed on an InfiniteM200 Pro Multimode Reader (TECAN, Seestrasse, Männedorf, Switzerland). Relative $fold change$ in luciferase activity was estimated, following normalization to Renilla luciferase activity.

CHO cells were cultured in F12 medium (Hyclone) supplemented with 10% FBS (Gibco) and Pen-Strep antibiotics (Hyclone) at 37 °C in 5% CO₂. Commercial pDisplay plasmid with C-myc tag at the C-terminal of the cloned gene of interest was transfected into CHO mammalian cells using Lipofectamine LTX & Plus reagent (Invitrogen) in 6 wells plates according to manufacturer’s protocol. 24 hr post transfection, medium was replaced with fresh medium including G418 drug (1.8 mg/ml) (Hyclone) to select for transfectants. Subsequently, the cells were expanded and subcloned by single cell dilution. Clones were then validated by IFA in 96 wells plate as follow: cells were fixed with 1% paraformaldehyde, 15 min at 37 °C and washed with PBS. The cells were then incubated for 1hr at 37 °C with chicken anti-C-myc antibody (Abcam) or rat anti-pooled CIR antibodies and/or rat preimmune serum (1:10 diluted in PBS 3% BSA). After washing 3 times with PBS, secondary Alexa 594 goat anti-chicken (ImmunoJackson) or Alexa 488 goat anti-rat (Biolegend) (1:600 and 1:500 dilution respectively in PBS 3% BSA) was incubated for 1hr at 37 °C. Nuclei was stained with Hoechst 33342 (1uM in PBS). The fluorescence signal was quantified using the fluorescence plate reader, Infinite M200 PRO multimode reader (TECAN).

The concentration of reduced (GSH) and oxidized (GSSG) glutathione, as well as the ratio between GSSG/GSH, was determined in monocytes’ samples using OxiSelect™ assay (STA-312; Cell Biolabs, San Diego, United States) according to the manufacturer’s instructions. An infinite M200 PRO multimode reader (TECAN, Männedorf, Switzerland) was utilized for recording the absorbance. Level of glutathione components, intermediates in the glutathione synthesis, and molecules involved in glutathione components: L-cysteine, L-glutamic acid, L-glycine, γ-L-glutamyl-L-cysteine, L-cysteinyl-L-glycine, 5-oxo-L-proline, and L-ornithine, were determined using GC-MS analysis. GPX3 was quantified using the MRM approach as described above.