Elx405

As previously carried out (Grimley et al., 2017 (link)), adherent RPMI 1640 (Gibco) cultured RCC or SKOV-3 cells were dissociated from tissue culture vessels and counted using the Countessä II. Diluted cells were then seeded at 500 cells/well in 96-well plates and allowed to adhere overnight during incubation. The next day, increasing concentrations of either screening compounds or DMSO were added to the test plates and incubated overnight. The following day the ELx405 (BioTek) microplate washer was used to lower the volume of culture medium and an equal volume of CellTiter-Glo (Promega) was added directly to the remaining culture medium in each well using a Multidrop Combi dispenser. To further induced cell lysis, test plates were allowed to shake on the Multidrop Combi dispenser plate carrier for 2 minutes following CellTiter-Glo^Ò addition. The PHEARstar (BMG Labtech) luminometer was used to measure luminescence, which is proportional to ATP levels (a measure of metabolically active cells).

Analysis of inflammatory cytokines in the supernatant of HUVEC cell cultures treated with Dox ± fidarestat was performed by using a human cytokine/chemokine magnetic bead multiplex kit from Millipore (#HCYTOMAG-60K) following manufacturer’s instructions. Briefly, after 24h of treatment, the culture media was collected and cleared by centrifugation. The media was concentrated (10x) using a vacuum evaporator, and the protein content was quantified using Bio-Rad protein assay (#500–0006). Equal amount of media was incubated with the pre-mixed beads with agitation on a plate shaker overnight at 2–8 °C. After the incubation, cells were washed with wash buffer using an automated magnetic plate washer (ELx405; Biotek) and incubated with detection antibodies for 1 h at room temperature. The wells containing detection antibodies were counterstained with Streptavidin-Phycoerythrin, incubated for another 30 min, washed, 150 μL of sheath fluid was added and analyzed with a Milliplex analyzer (Millipore). The results are expressed as pg/mL based on the standard curve generated with the standards provided with the kit using Luminex xPONENT software. Similarly as described above, cytokine levels in the mice aorta tissue homogenates were measured using mouse inflammatory multiplex kit from Millipore (#MCYTOMAG-70K).

The p24 Luminex capture assay was performed in a flat bottom 96-well plate. The washes were performed using an automatic magnetic washer; and the plates were kept from the light during incubation. The magnetic beads were diluted in the assay buffer (20mM Tris Hcl pH6, 0.1% BSA and 0.05% Tween 20) to a concentration of 60,000 beads/ml and 50μl (3000 beads) were added in each well. The samples and the p24 standards were lysed by adding a 10% volume of a 10X lysis solution (10X Triton X-100 and 0.05% Tween 20) for 1 hour at 37°C, and then diluted in assay buffer. 50μl of the lysed samples or standard were added to the magnetic beads and incubated for 1 hour at RT under agitation. The plates were then washed twice with 200μl of a wash solution (20mM Tris Hcl pH6 and 0.05% Tween 20) using a magnetic microplate washer (ELx405, BioTek, Winooski, VT, USA). 100μl of the detection antibody anti-p24 KC57 (Beckman Coulter, Fullerton, CA, USA) was added to each well, and the plates were incubated for 1 hour at RT under agitation. The plates were washed twice with 200μl of a wash solution. The magnetic beads were resuspended in 100μl of assay buffer and agitated for 10 minutes before the analysis. The analysis of the magnetic beads was done on a Luminex 100 using the Bioplex manager software version 6.0 (Bio-Rad, Hercules, CA) and a minimum of 300 beads per well were analyzed.