Effects Of THP-1 Cell Culture

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THP-1 cells and THP-1 cell culture methodology were also provided by Ondek Pty Ltd. THP-1 cells were cultured in RPMI-10 culture medium. The first THP-1 cell culture was made by Dr Senta Walton, with a starting concentration of 2 x 105 cells/mL. The cell culture was then incubated at 37°C with 5% CO2 and subcultured every 2-3 days, when the cell concentration reached approximately 8 x 105 cells/mL.
THP-1 cell based assay
THP-1 cell based assay was performed to determine the immune modulatory effects of H. pylori and its derived products. In this assay, THP-1 cells were stimulated by 0.5 µg/mL (0.1 µg/well) Escherichia coli LPS, exposed to different H. pylori products at various product concentrations, and incubated for 22 hours. Activation
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pylori product solutions were diluted to various concentrations. Each H. pylori product was assessed over eight concentrations. Samples were diluted in two-fold serial dilution steps starting with 100 µg dry weight per well. H. pylori product samples were prepared in PBS.
LPS solution was made at a concentration of 2µg/mL in RPMI-2HS.
Assay was composed of negative controls, positive controls, and test samples. Negative controls in the assay contained no LPS stimuli and no H. pylori product. Positive controls contained LPS stimuli without H. pylori product. Product samples contained H. pylori products at various concentrations and LPS stimuli. In separate wells, THP-1 cells were also exposed to H. pylori and its derived products without LPS stimulation to assess the immune activatory properties of H. pylori products. Each sample was assessed in triplicate.
Assay components were added to the 96-well plate in a specific order to reduce cross-contamination and accidental activation of THP-1 cells. Assay components were added to the wells in the following order: 100 µL THP-1 cells (2 x 105 cells/well) was added to every
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A volume of 48 µL antibody master mixture solution containing 0.5 µL CD80 and 0.01 µL viability stain in FACS buffer was added, and the samples were thoroughly mixed. The plates were incubated for 20 minutes in the dark at room temperature. After that, 130 µL of FACS buffer was added to each well, followed by centrifugation. The supernatant was discarded and the well content was resuspended with 150 µL of FACS buffer, which was again followed by centrifugation. The supernatant was discarded and the well content was resuspended in stabilising fixative. The plates were covered with aluminium foil and stored at 4°C until sample acquisition. Filtration using nylon mesh was done for turbid samples, particularly for live and heat-killed samples, prior to acquisition.
Assay acquisition by flow cytometry
Assay acquisition by flow cytometry was performed in LSRII Fortessa flow cytometry instrument at the CMCA facility. Dr. Senta Walton performed the calibration of the cytometer as well as the instrument setup and was responsible for sample acquisition. Flow cytometry data acquisition was closely observed to make sure that there was no blockage.

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