Spectramax abs plus microplate reader

The trypanosome inhibition assay was performed using the Alamar blue assay as described by Raz [68 (link)] and Pimentel-Elardo [52 (link)], with minor modifications. Bloodstream form Trypanosoma brucei brucei cells (Isolate STIB247, [76 (link)]) were cultured in Hirumi’s Modified Iscove’s (HMI-9) medium with the modifications that foetal bovine serum (FBS) was increased to 15% (v/v) and serum Plus^TM was omitted [77 (link)].
A defined number of parasites, 1 × 10⁵ trypanosomes mL⁻¹ (5 µL), were seeded in each well (Corning™ clear polystyrene 96-well sterile microplate) containing HMI-9 medium and the test compound (previously dissolved in 2% sterile DMSO) (100 µL). Alamar blue (resazurin sodium salt, 0.05 mg/mL, Sigma) was added to the wells. Assays were performed in triplicate and incubated for 20−23 hrs at 37 °C in an atmosphere of 5.0% CO₂. Staurosporine (Sigma) was used as the reference antibiotic. The positive control consisted of the trypanosomes in culture medium with 2% DMSO, while the negative control was comprised of the medium without the trypanosomes. The absorbance was measured on a SpectraMax ABS Plus microplate reader (Molecular Devices, Wokingham, UK) at a wavelength of 570 nm and reference wavelength of 600 nm.

Cell viability of the prepared hydrogels was evaluated for toxicity of human embryonic kidney 293 (HEK-293) cells using WST-8 (QuantiMax, Seoul, Republic of Korea) assay. HEK-293 cells were seeded at 2 × 10⁴ per well in a 96-well plate loaded with hydrogel samples. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, WELGENE, Gyeongsan-si, Republic of Korea) containing 10% fetal bovine (FBS) and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin). The plate was then incubated at 37 °C with 5% CO₂ air for 48 h. After that, the hydrogels and the culture supernatant were removed, and a new DMEM and WST-8 solution were added and incubated for 4 h. The optical density was determined at a wavelength of 450 nm with a SpectraMax ABS Plus Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The cell viability was determined using the following equation [31 (link)]: $$Cell viability \left(\%\right)=\left(A_{samples}/A_{Control}\right)\times 100\%$$

A biofilm assay was conducted in the form of a preliminary study after selecting 10 intraventricular and 10 lumbar intrathecal CSF samples from the total number of cases. After overnight culture at 37 °C, the culture broth in the 96-well plate was discarded and residual liquid was further removed by tapping the plate upside down on several layers of paper towel. Biofilms attached inside each well were washed three times with 200 µL of phosphate buffered saline (PBS, pH 7.2) and at every washing the residual liquid was removed by tapping on paper towels. After washing, biofilms in the plate were stained with 100 μL of 0.1% (w/v) crystal violet solution for 10 min at room temperature. After discarding the crystal violet solution, microtiter plate wells were washed three times with 200 μL of double distilled water and air-dried for 1 h. Finally, 200 μL of 95% ethanol was added into each well and crystal violet was eluted from the biofilms by pipetting followed by incubation at room temperature for 15 min. After another pipetting to mix the solutions well, the optical density of the eluted dye solution was determined using a SpectraMax ABS Plus Microplate Reader (Molecular Devices, LLC., San Jose, CA, USA) at 550 nm.

The cells with or without LPS stimulation were harvested after 6 h. TRI reagent™ (Invitrogen, Waltham, MA, USA) was used to extract total RNA, and 100% isopropanol was used to precipitate RNA. RNA was collected via centrifugation at 13,000 rpm and 4 °C for 10 min. The RNA pellet obtained was washed with 70% EtOH. After drying, the total RNA pellet was resuspended in 20 µL of nuclease-free water. Total RNA was quantified using a SpectraMax^® ABS Plus Microplate Reader (Molecular Devices, San Jose, CA, USA), and cDNA was synthesized using 500 ng of this RNA and a Power cDNA Synthesis Kit (Intron Biotechnology, Seongnam-Si, Korea), according to manufacturer’s instructions.

Total antioxidant capacity (TAC) in the liver was measured using a commercial kit (QuantiCromAntioxidant Assay Kit; DTAC-100) (BioAssay Systems, Hayward, CA, USA). Approximately 100 mg of frozen liver samples were homogenized in 1 mL of PBS for 45 s using a beads beater and centrifuged at 10,000× g for 10 min. Aliquots of supernatants were taken for the analyses of protein content using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Cleveland, OH, USA) after 20 times dilution. Afterwards, TAC was measured according to the manufacture’s protocol without further dilutions. The absorbance was measured using SpectraMax^® ABS Plus microplate reader (Molecular devices, San Jose, CA, USA). The TAC values were expressed as nM trolox quivalents/mg protein.

The levels of nitrite, a stable oxidized product of NO, were measured in the culture media using the Griess reagent. Both serum nitrite and nitrate (NOx) concentration were determined with a nitrate reductase-based colorimetric assay kit (Alexis, Los Angeles, CA, USA). Triplicates of each sample were incubated with the same volume of sulfanilamide and N-(1-Naphthyl) ethylenediamine solution. After 5–10 min, absorbance levels were measured at 550 nm using a SpectraMax ABS Plus microplate reader (Molecular Devices, San Jose, CA, USA).