PANC-1 and MIA PaCa2 cells were seeded in black, clear-bottom 96-well plates (Corning) in DMEM without serum. The following day, macrophage conditioned media were added in a 1:1 dilution with complete DMEM. BrdU labeling solution was given to cells after 48 h of conditioned media treatment. All experimental steps in the BrdU assay were performed according to the manufacturer’s instructions (Chemiluminescent BrdU assay, Roche). Luminescence was measured on a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT). For testing the functional importance of TNF-α in M0 conditioned medium, we included human recombinant TNF-α (Sigma) in increasing concentrations from 10 pg to 10 ng and the TNF-α inhibitor Infliximab (Remicade, MSD, Kenilworth, NJ) at 25 μg/ml.
Chemiluminescent brdu assay
Manufactured by Roche
The Chemiluminescent BrdU assay is a laboratory equipment product that measures the incorporation of the thymidine analog bromodeoxyuridine (BrdU) into cellular DNA. This assay utilizes a chemiluminescent detection method to quantify the amount of BrdU present in the sample, providing information about cell proliferation.
Automatically generated - may contain errors
4 protocols using chemiluminescent brdu assay
1
Macrophage-Mediated Pancreatic Cancer Proliferation
2
Macrophage-Mediated Pancreatic Cancer Proliferation
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PANC-1 and MIA PaCa2 cells were seeded in black, clear-bottom 96-well plates (Corning) in DMEM without serum. The following day, macrophage conditioned media were added in a 1:1 dilution with complete DMEM. BrdU labeling solution was given to cells after 48 hours of conditioned media treatment.
The BrdU assay was performed according to the manufacturer's instructions (Chemiluminescent BrdU assay, Roche). Luminescence was measured on a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT). For testing the functional importance of TNF-a in M0 conditioned medium, we included human recombinant TNF-a (Sigma) in concentrations from 10 pg to 10 ng and the TNF-α inhibitor In iximab (Remicade, MSD, Kenilworth, NJ) at 25 ug/ml.
The BrdU assay was performed according to the manufacturer's instructions (Chemiluminescent BrdU assay, Roche). Luminescence was measured on a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT). For testing the functional importance of TNF-a in M0 conditioned medium, we included human recombinant TNF-a (Sigma) in concentrations from 10 pg to 10 ng and the TNF-α inhibitor In iximab (Remicade, MSD, Kenilworth, NJ) at 25 ug/ml.
3
Evaluating Macrophage Influence on Pancreatic Cancer Cell Proliferation
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PANC-1 and MIA PaCa2 cells were seeded in black, clear-bottom 96-well plates (Corning) in DMEM without serum. The following day, macrophage media (M0, M1, MM2, and TAM) were added in a 1:1 dilution with complete DMEM. BrdU labeling solution was given to cells after 48 hours of conditioned media treatment. The BrdU assay was performed according to the manufacturer's instructions (Chemiluminescent BrdU assay, Roche). Luminescence was measured on a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT). For testing the functional importance of TNF-α in M0 conditioned medium, we included the commercially available TNF-α inhibitor in iximab (Remicade, MSD, Kenilworth, NJ) at 25 ug/ml.
4
Macrophage Conditioned Media Impact on Pancreatic Cancer Cell Proliferation
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PANC-1 and MIA PaCa2 cells were seeded in black, clear-bottom 96-well plates (Corning) in DMEM without serum. The following day, macrophage conditioned media were added in a 1:1 dilution with complete DMEM. BrdU labeling solution was given to cells after 48 hours of conditioned media treatment. All experimental steps in the BrdU assay were performed according to the manufacturer's instructions (Chemiluminescent BrdU assay, Roche). Luminescence was measured on a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT). For testing the functional importance of TNF-α in M0 conditioned medium, we included human recombinant TNF-α (Sigma) in increasing concentrations from 10 pg to 10 ng and the TNF-α inhibitor In iximab (Remicade, MSD, Kenilworth, NJ) at 25 ug/ml.
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