Synergy mx spectrophotometer

MDA-MB-231 breast cancer cells (ATCC HTB-26) were maintained in culture in phenol-red free L-15 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 12.5 μg/mL amphotericin (PSA) at 37 °C in a humidified incubator. HeLa cervical cancer cells (ATCC CCL-2) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented as above at 37 °C and 9% CO₂ in a humidified incubator.
The antiproliferative effects of the compounds were assessed using the WST-1 assay (Sigma-Aldrich, Johannesburg, South Africa) as previously described [35 (link),36 (link)]. Briefly, cells were seeded at a density of 6000 cells per well into 96-well plates and incubated overnight, followed by treatment with a range of concentrations (0.32, 1.6, 8, 40, 200 and 1000 μM) of the compounds or dimethyl sulfoxide (DMSO) vehicle control (0.02% v/v DMSO) for 96 h. Thereafter 2.5 µL of WST-1 Cell Proliferation Reagent was added per well and the absorbance at 450 nm after 8 h recorded using a Synergy Mx spectrophotometer (BioTek). The half maximal inhibitory concentration (IC₅₀) for each compound was calculated relative to the vehicle-treated control from a dose response curve (log concentration vs absorbance at 595 nm) using non-linear regression with GraphPad Prism 4 (GraphPad Inc., San Diego, CA, USA).

The LOQ is the lowest analyte DNA concentration that provides an acceptable level of precision (i.e., 3/3 replicates amplified), whereas LOD is defined as the concentration of a measurand that is significantly different from a negative control (i.e., at least 2/3 replicates amplified) [28 (link)]. LOD and LOQ were measured through the qPCR and LAMP assays using tenfold serial dilutions of B. cenocepacia AU1054 and B. cenocepacia J2315 genomic DNA. LAMP products were directly analyzed with both the naked eye, and 2% agarose gel electrophoresis. Furthermore, the absorbance in LAMP products was measured using a Synergy MX spectrophotometer from BioTek Instruments, Inc. (Winooski, VT, USA) at 434 and 560 nm [19 (link)]. A reaction was considered positive when the color value was above 0.05 (ΔOD = OD_434nm − OD_560nm; difference in absorbance of samples at 434 (increased absorbance) and 560 nm (decreased absorbance)). Negative reactions (i.e., wells with ΔOD values below 0.05) were analyzed by 2% agarose gel electrophoresis to confirm if a result is a true negative.

DCs were isolated according to the protocol described above. Between days 8 and 10 of cultivation, cells were collected for co-culturing. GL261 tumor cells were stimulated as described above and incubated for 24 h. The cells were then subjected to six cycles of freezing (–80 °C) and thawing (+55 °C). Total protein in the cell lysate was measured with a commercial BCA Protein Assay Kit (Sigma-Aldrich) and a Synergy MX spectrophotometer (BioTek Instruments Inc., USA). Two mg of protein was added to a suspension of 10 × 10⁶ DCs for 90 min. To activate the DCs, they were treated with lipopolysaccharide (0.5 μg/ml) for 24 h. In some experiments, PBS or DCs co-cultured with GL261 glioma cell lysates subjected to several freeze/thaw cycles to induce accidental necrosis (without photoinduction) were used as controls.

Serum urea levels were evaluated through colorimetric assays based on the urease method using deproteinized serum samples (Labtest, Brazil). Values were determined using a Synergy Mx spectrophotometer (Biotek, Winooski, Vermont, USA) and analyzed accordingly.

The chemical structure of SF and LPA/SF was analyzed using ATR-FTIR (Perkin Elmer, Waltham, MA, USA) at the spectra wavelength range of 4000–400 cm⁻¹.
The morphology was investigated by field emission scanning electron microscopy (FESEM, Hitachi S4700) and atomic force microscopy (AFM) on a Scanning Probe Microscope XE 70 (Multimode-8, Bruker, Billerica, MA, USA). Contact angle characterization was carried out by employing water contact goniometer (TantecTM, CAM-PLUS Micro, Schaumburg, IL, USA) to measure the hydrophilicity of tissue culture polystyrene (TCP), SF, and LPA/SF. Transparency of SF and LPA/SF was evaluated by SYNERGY Mx spectrophotometer (BioTek, Winooski, VT, USA) at a wavelength range of 380 nm–780 nm after immersing in PBS.