CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 76 AFRICA Group 1: the normoxic control group represented untreated cells that were maintained in their normal environment in the standard 5% CO2 incubator for the period of the experiment. Group 2: the SI control group for the pre-ischaemia inhibitorexposed groups consisted of cells that were incubated for seven hours in the hypoxic incubator, followed by incubation with 1% HS DMEM for one hour in the normoxic incubator. Group 3: cells were treated with TNF (recombinant murine TNF, Peprotech, USA, 0.5 ng/ml in DMEM with 1% HS) for 30 minutes in the normoxic incubator. Cells were then re-incubated with fresh 1% HS DMEM for 60 minutes in the normoxic incubator before incubation with SI buffer for seven hours in the hypoxic buffer. Finally, cells were incubated with 1% HS DMEM for one hour in the normoxic incubator. Group 4: ammonium pyrrolidone derivative dithiocarbamate (PDTC) (100 µM)28 (Sigma-Aldrich, USA) was added prior to the addition of TNF (0.5 ng/ml) in 1% HS DMEM and incubated in the normoxic incubator. Following re-incubation for 60 minutes with 1% HS DMEM in the normoxic incubator, cells were incubated for seven hours in the hypoxic incubator. Cells were then incubated in 1% HS DMEM for one hour in the normoxic incubator. Group 5: PDTC was added for 30 minutes in 1% HS DMEM followed by two washes, and incubated in 1% HS DMEM for one hour in the normoxic incubator. Following re-incubation for 60 minutes with 1% HS DMEM in the normoxic incubator, cells were incubated for seven hours in the hypoxic incubator. Cells were then incubated in 1% HS DMEM for one hour in the normoxic incubator. Group 6: PDTC (100 µM) was added for one hour after the seven-hour SI with TNF preconditioning. Group 7: PDTC was added for 30 minutes in 1% HS DMEM, followed by two washes, and incubated in 1% HS DMEM for one hour after the seven-hour SI. In all the groups, cells were washed twice with PBS (pH 7.4) prior to the change of medium. Cell viability Cell viability was evaluated using the trypan blue exclusion method.29 Cells were trypsinised and resuspended in PBS. A small volume of cell suspension was mixed in a 1.1 ratio with trypan blue (4 µM) (Sigma, Germany). The trypan blue seeps through the disrupted membrane of dead or dying cells and in the process, stains them blue. The mixture is pipetted onto a Neubauer haemocytometer and the counting is assessed on a light microscope. The ratio of blue-stained cells to grey, unstained live cells is calculated and expressed as a percentage. Respiratory parameters Respiration studies were conducted immediately after the simulated index ischaemia (Fig. 2). State-2 respiration was measured using an Oxytherm respirometer equipped with a Clark-type electrode and a Peltier temperature-control unit (Oxytherm, Hansatech, Norfolk, UK). Calibration was set with PBS at a temperature of 37°C. Calibration was verified by the addition of 1 ml PBS into the chamber followed by a baseline reading. The percentage viability was used to calculate a factor from which the respiration values from viable cells could be determined. Protein isolation To investigate the role of NFκB as a trigger, PDTC was administered during the ischaemic or TNF-preconditioning stimulus. Samples were collected after 15 minutes as this timepoint was observed as the peak of IkB phosphorylation (data not shown). After exposure to the drugs, the cells were washed quickly with PBS (pH 7.4) and then transferred to the respective Eppendorf in the presence of 500 µl solution of lysis buffer containing 10% Nonidet P-40, 4 M NaCl, 1 M Hepes (pH 7.9), 500 mM EDTA and complete EDTA-free protease inhibitor cocktail (Roche Systems, USA). Samples were centrifuged at 3 000 rpm for 30 seconds and the supernatant was transferred to a new tube. These tubes were spun for five minutes at 5 000 rpm. The subsequent supernatant fraction contained the cytosolic proteins. Protein concentrations were determined using Lowry’s protein method.30 Extracts were stored at –80°C. Bio-plex array analysis for phosphoprotein testing A singleplex assay was performed to test for phosphorylated IkB proteins from cell lysates. The Bio-plex phosphorylation assay and testing reagent kits (Bio-Rad Laboratories Inc, USA) were used according to the manufacturer’s instructions. Bio-plex phosphoprotein assays are bead-based singleplex or multiplex assays that detect phosphorylation of proteins in cell and tissue sample lysates. The lysates are coupled to internally dyed beads and incubated with the specific biotinylated detection antibody in a 96-well plate. Streptavidin-phycoerythrin (streptavidin-PE) is then added to bind the detection antibodies. Data are acquired using a dual laser, flow-based microplate reader system and outputted as fluorescence intensity on the Bio-plex Manager™ software. CTL TNF TNF + PDTC Phospho Ikk activity (AU) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 ### Fig. 2. Effect of NFκB inhibitor (PDTC) on the phosphorylation of IkB during the preconditioning stimulus in C2C12 myotubes subjected to TNF-mediated preconditioning. The addition of PDTC (100 µM) reduced the phosphorylation of IkB observed during the TNF preconditioning stimulus. *p < 0.05 vs ischaemic control (CTL), ##p < 0.01 vs TNF; n ≤ 4 per group. AU: arbitrary units.
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