Varioskan multimode plate reader

In order to rule out cytotoxic side effects of the biologically active cycloheximide derivatives on eukaryotic cells, the viability of THP-1 cells was measured using the Alamar Blue reagent (Biozol, #BZL00726) according to manufacturer’s instructions. Briefly, THP-1 cells were prepared as described above. Instead of infecting the cells, they were incubated with 100 μM MT_30.32, or MT_30.51 for 20 h, at which time point Alamar Blue reagent was added. After another 4 h, cell viability was assessed by measuring fluorescence intensity at 590 nm (excitation 570 nm) using a Varioskan™ Multimode plate reader (Thermo Scientific).

Endogenous ROS generation in platelets was determined according to the method of Thushara et al.51 (link). WP (5 × 10⁶ cells/mL) were taken separately in 96-well plates and treated with A23187 (1 μM) as positive control or UCB (0–200 μM) as test and the final volume was made up to 200 μL with Tyrode’s buffer and incubated for 30 min at 37 °C. For inhibition studies, pre-loaded platelets with UCB (200 μM), were incubated with Mito-TEMPO (10 μM) and incubated for 30 min at 37 °C. The untreated and treated platelets were then incubated with CM-H2DCFDA (10 μM) for 30 min at 37 °C and the fluorescence was recorded using a Varioskan multimode plate reader (Thermo Scientific, USA) by exciting the samples at 488 nm and measuring the resulting fluorescence at 530 nm. The same procedure was followed for untreated WP obtained from HS, HB and experimental animals.

Mural granulosa cells from follicular fluid were homogenized in a RIPA buffer pH 7.4 (BioBasic, Toronto, Canada) including Phosphatase and Protease inhibitors (Roche, Basel, Switzerland). After homogenization protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA), and a Varioskan multimode plate reader (Thermo Scientific). Thirty micrograms of total protein were loaded to a Criterion XT precast gel (4–20% bis–tris) (Biorad, CA, USA). Proteins were subsequently transferred to a nitrocellulose membrane and incubated with anti-FMRpolyG 8FM and 9FM (1:10,000) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, England. Ab8245; 1:10,00, mouse monoclonal) as internal loading control. The membrane was scanned with ChemiDoc™ XRS+ imager and the intensity of the bands of interest was quantified using Image Lab software (Biorad, CA, USA).

Antitrypanosomal screening of the crude extracts, and later of the fractions of
the TMs, was conducted using the Alamar blue assay [41 (link)] with slight modifications. Serial
dilutions of the extracts were prepared on a 96-well plate with a starting
concentration of 100 μg/mL to 0.1953 μg/mL for all samples, except for the
positive control which had a starting concentration of 25 μg/mL to 0.0488 μg/mL.
The final concentration of DMSO in the well with the highest percentage of DMSO
was 0.5%. The cell density of trypanosomes in their logarithmic growth phase was
adjusted to 2 x 10⁵ cells/ml and 100 μL of this suspension was added
into each well, excluding the media control well to which no cell was added. The
negative control consisted of only trypanosome cells in media with no treatment
(extracts, fractions, or drug) added. Diminazene aceturate (DA), a standard
antitrypanosomal drug, served as the positive control. After 24 hours
incubation, 20 μL of 500 μM Alamar blue dye was added and incubated for an
additional 24 hours. Fluorescence readings were taken using a Varioskan
multimode plate reader (ThermoFisher Scientific, USA) at an excitation
wavelength of 530 nm and emission wavelength of 590 nm. The experiment was
carried out in three biological replicates, with each biological replicate
containing three technical replicates.

Transfected COV434 cells were harvested and homogenized with a RIPA buffer, pH 7.4 (BioBasic, Toronto, Canada) containing phosphatase and protease inhibitors (Roche, Basel, Switzerland). A Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was to calculate protein concentrations, and plates were read in a Varioskan multimode plate reader (Thermo Scientific). Criterion XT precast gels (4–20% bis–tris) (Biorad, Hercules, CA, USA) were loaded with 50 µg of total protein per well and transferred to a nitrocellulose membrane. Primary antibodies were anti-AMH (Ab229212, 1:2000, Abcam) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Ab8245; 1:10,000, mouse monoclonal, RRID: AB2107448, Abcam). ChemiDocTM XRS + imager was used to scan the membrane, and Image Lab software (Biorad) assisted in quantifying the intensity of the bands of interest.

Rat heart mitochondria were isolated from LV tissues as previously described^{27 (link)}. Isolated mitochondria were resuspended in buffer containing 250 mM sucrose, 20 mM Tris–HCl, and 40 mM KCl^{9 (link),10 (link)}. Then, 200 µg of isolated mitochondria were loaded with 5 µM membrane-permeable fluorescent probe, MitoSOX Red (Invitrogen, USA), in the dark for 15 min at 37 °C^{28 (link)} in the presence of 5 mM glutamate and 5 mM malate as complex I respiratory substrates. Fluorescence intensity was measured using Varioskan multimode plate reader (Thermo Scientific, USA) at 510 nm excitation and 580 nm emission wavelengths and was expressed relative to the vehicle control group.

Cells were seeded at 8×10³ cells in 96-well plates. After 24 h, CellTiter-Glo reagent was added at a 1:1 volume ratio to cell culture medium and incubated at room temperature for 10 min. The luminescence signal was analyzed with a Varioskan multimode plate reader (Thermo Scientific).