Synergy htx multi reader

Supernatants were obtained from cells exposed to the different treatments for a 24-h period. Treatments consisted of control (cells + DMSO), butein-treated, TNF-α-stimulated, and co-treated (butein (5 μM) + TNF-α (40 ng/ml) TNBC cells were collected and centrifuged at 1000 rpm for 4 min at 4°C. Specific ELISA assays for CCL2 (MCP-1) was performed following the manufacturer’s instructions. Shortly, 100 μl of supernatants from each sample and standards were added to 96 well plates pre-coated with capture antibody and incubated for 2.5 h at room temperature under shaking. After washing, 100 μl of prepared biotinylated antibody mixture was added to each well and incubated for 1 h. The mixture was decanted, and 100 μl streptavidin solution was added to each well and incubated for 45 min. Substrate reagent (100 μl) was then pipetted into each well and incubated for 30 min, followed by the addition of 50 μl of stop solution. Samples were assayed at an optical density of 450 nm using Synergy HTX Multi-Reader (BioTek, USA).

The effects of PL on cell proliferation were determined for MM-231 and MM-468 TNBC cells based on the dose-response viability study concentrations [92 (link)] using Alamar Blue^®. Briefly, cells were plated at an initial density of 5×10³ cell/well in 96-well plates and incubated overnight. The cells were treated for 72 or 96 h with PL at concentrations ranging between 0–50 μM or 0–10 μM in MM-231 or MM-468 cells, respectively, in a final volume of 200 μL/well. Control cells were exposed to DMSO at a concentration < 0.1%. Taxol^® was used as a positive control only at 1 μM concentration level [93 (link)], and equivalent wells without cells were used as a blank. Proliferation was measured after the predesigned experimental period by adding Alamar Blue^® to each well at 10% v/v and incubating for an additional 4 h. The plates were read at an excitation/emission of 530/590 nm using a Synergy HTX Multi-Reader (BioTek, USA).

In this experiment, cells were incubated overnight in the experimental media at a density of 5×10⁴ cell/well in 96-well plates. Both types of cells were treated for 24 h with TNF-α (0–100 ng/mL) or PL (concentration ranges of 0–50 μM in MM-231 or 0–10 μM in MM-468 cells). PL was solubilized in DMSO, while TNF-α was dissolved in cell culture water. Control wells were treated with DMSO at the highest used concentration (< 0.1%). Equivalently treated wells without cells were used as blanks. In this assay, Alamar Blue^® was used to determine cell viability at a concentration level of 10% v/v and an incubation time up to 6 h. The fluorescent fuchsia—reduced resazurin dye was measured at an excitation/emission of 530/590 nm using a Synergy HTX Multi-Reader (BioTek, USA).

PGG effect on GRO and GRO-α expression was investigated using individual quantitative ELISA assays. Supernatants from the control (cells + DMSO), PGG-treated (6.25- MM-231 and 25 µM- MM-468), TNF-αstimulated (50 ng/ml), and co-treated (PGG + TNF-α) cells were collected, and the cells centrifuged at 1000 rpm for 4 min. Experiments were performed in triplicate according. Supernatants from samples and standards (100 μl) were pipetted into the 96 well plates and incubated at room temperature. After 2.5 h, the plates were washed, and the biotinylated antibody was added. Following 1 h, streptavidin (100 μl) was added, and within 45 min of incubation, the substrate reagent was incubated for 30 min. The quantified data was determined by optical density at 450 nm (Synergy HTX multi-Reader- BioTek, USA).

MDA-MB-231 and MDA-MB-468 TNBC cells were subjected to various treatments for 24 h and had their supernatants collected. The following treatments were used: control (cells only), TQ (75 μM), TNF-α (50 ng/mL), and TQ (10 μM) + TNF-α (50 ng/mL). The Raybiotech ELISA CCL2, CCL3, CCL4, and CCL20 kit instructions were used for the experiment. TNBC cells were obtained and centrifuged for 4 min at 4 degrees Celsius at 1000 rpm. According to the manufacturer’s recommendations, a particular ELISA for the detection of human CCL2, CCL3, CCL4, and CCL20 was used. In brief, 100 μL of the supernatants from each standard and sample were put into 96-well plates that were precoated with capture antibody, and they were then incubated for 2.5 h at room temperature while being shaken. After washing, a 100 μL combination of biotinylated antibodies was made, poured into each well, and allowed to sit for one hour. The mixture was then aspirated, and 100 μL of streptavidin solution was added to each well before it was incubated for 45 min. After adding 50 μL of stop solution, 100 μL of substrate reagent was pipetted into each well and incubated for 30 min. The optical density of the samples was determined at 450 nm using a Synergy HTX Multi-Reader (BioTek, Winooski, VT, USA).