Synergy htx

The samples were placed in 96 well microtitration plate (Nunc™ MicroWell™, Thermofisher Scientific, Waltham, USA). All measurements were performed at λ = 455 nm, 22° C on the Synergy HTX microplate reader (Synergy HTX, BioTek, Vermont, USA).

An overnight culture of C. rodentium was grown in LB media at 37 °C with shaking. The culture was diluted 1:100 in LB before compounds were added at the concentrations listed and a vehicle was used as a control. 0.5-mL culture replicates of 5 were incubated for 6 h in CytoOne 96-well flat-bottom plates with the lid at 37 °C, with shaking in the BioTek Synergy HTX plate reader and incubator. Cell density was measured as OD₆₀₀ after 6 h with the BioTek Synergy HTX plate reader and incubator (Winooski, VT, USA), beginning at the time of dilution.

Levels of interleukin (IL)-8, monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor alpha (TNF-α) were measured by ELISA 24 h post-treatment (R&D Systems, Minneapolis, MN, USA). Culture supernatants containing HIV particles were used to measure p24 protein levels by ELISA 24 h post-treatment according to the manufacturer’s protocol (RETRO-TEK). The O.D. was read at A450 on a Synergy HTX plate reader (BioTek). In addition, protein lysates from human astrocytes after 8 h treatment were used to measure NF-κB, c-Jun N-terminal kinase (JNK) and p38/MAPK protein expression by ELISA (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. The O.D. was read at A450 on a Synergy HTX plate reader (BioTek).

Samples were placed in 96-well microtitration plates (Nunc™ MicroWell™ 96-Well Microplates, Thermofisher Scientific, USA). Measurements were performed at 22°C on the Synergy HTX microplate reader (Synergy HTX, Biotek USA).

OCN secretion by hAMSCs was evaluated at days 14
and 21 of osteogenic
differentiation using in vitro SimpleStep ELISA (enzyme-linked
immunoabsorbent assay) kits for human OCN (ab270202, Abcam) and human
OPN (ab269374, Abcam), respectively, according to the manufacturer’s
instructions. OCN levels were quantified by measuring the absorbance
at λ = 450 nm in a microplate reader (Synergy HTX, Biotek Instruments)
and were expressed in nanograms of protein, normalized to dsDNA content.
(As previously mentioned, OPN quantification produced too low and
variable results, hindering their discussion.) Regarding calcium quantification,
20 μL of each sample (cell lysates from days 7, 14, and 21)
and each calcium standard (0, 4, 6, 8, and 10.0 mg/dL) was mixed with
20 μL of HCl (1 M) for 30 min at RT in a 96-well plate. In another
96-well plate, 20 μL of the previously prepared solutions were
added to 80 μL of the reagents provided in the QuantiChrom calcium
assay kit (DICA-500, BioAssay Systems), according to the instructions
of the manufacturer. Absorbance readouts were measured at λ
= 612 nm using a microplate reader (Synergy HTX, Biotek Instruments).
Calcium concentrations were normalized by the total dsDNA in each
sample.

hCECs (1 × 10⁴) were cultured in 96-well plates and treated with miRNA or IFN-γ for 48 h. Cell viability was measured using the Cell Counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The plates were incubated with CCK-8 solution for 1–2 h. Cell viability was determined by measuring the absorbance at 450 nm using a microplate spectrophotometer (Synergy HTX, BioTek, Winooski, VT, USA).
The cell proliferation rate was measured using a commercial bromodeoxyuridine (BrdU) proliferation assay kit (GmbH; Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. Cells (5 × 10³ cells/well) were seeded into 96-well plates. Cells were labeled with BrdU at 37 °C and 5% carbon dioxide (CO₂) for 48 h. After incubating the plate in the FixDent solution at 25 °C for 30 min, the cells were incubated with the anti-BrdU-POD solution at 25 °C for approximately 90 min. Then, the substrate solution was added to each well and the plate was incubated at 25 °C for 20 min. Thereafter, 1 M sulfuric acid (H₂SO₄) was added to each well to stop the reaction. Optical density was measured at 450 nm using a microplate reader (Synergy HTX, BioTek). Proliferation rates were expressed as the percentage of controls after subtraction of the corresponding blanks.

Cell viability was assessed using a tetrazolium-based assay using microplate reader (Biotek, SYNERGY HTX, Vermont, USA). IC₅₀ values were determined through the dose-response curves.
Cologenic survival was defined as the ability of the cells to form colonies. Images were taken and analyzed by microscopy (Leica, DFC450C, Wetzlar, Germany) and microplate reader (Biotek, SYNERGY HTX, Vermont, USA).