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 …show more content…
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 …show more content…
pylori, the CD80 gMFI values of THP-1 cells exposed to H. pylori derived products in the absence of LPS stimulation were determined and compared with the corresponding value of negative controls. Statistical analysis of the values was then performed using one way ANOVA with Tukey’s post-hoc test to find any statistically significant differences between the means of CD80 gMFI values of the negative control, positive control, and test samples. Samples able to activate THP-1 cells had a higher CD80 gMFI value compared with the negative control. One-way ANOVA with Dunnett’s post-hoc test was then performed to compare the CD80 gMFI value of each test sample with the gMFI value of wild type sample (or Student’s t-test when only 2 sample groups were included in the analysis). The result therefore provides information about the ability of a test sample to activate THP-1 cells as compared to the wild
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 …show more content…
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 …show more content…
pylori, the CD80 gMFI values of THP-1 cells exposed to H. pylori derived products in the absence of LPS stimulation were determined and compared with the corresponding value of negative controls. Statistical analysis of the values was then performed using one way ANOVA with Tukey’s post-hoc test to find any statistically significant differences between the means of CD80 gMFI values of the negative control, positive control, and test samples. Samples able to activate THP-1 cells had a higher CD80 gMFI value compared with the negative control. One-way ANOVA with Dunnett’s post-hoc test was then performed to compare the CD80 gMFI value of each test sample with the gMFI value of wild type sample (or Student’s t-test when only 2 sample groups were included in the analysis). The result therefore provides information about the ability of a test sample to activate THP-1 cells as compared to the wild