Upon sacrifice the peritoneum of mouse was perfused with 5 mL of PBS containing 1% FCS (GIBCO, ThermoFisher Scientific). The lavage was passed through a 70 um cell strainer (Corning, 431,751) and centrifuged for 10 min at 4°C at 350 x g. Cells were resuspended in culture medium (RPMI-1640 containing 11.1 mM glucose, 100 U/ml penicillin, 100 μg/mL streptomycin, 2 mM glutamax, 50 μg/mL gentamycin, 0.25 μg/mL Amphotericin B and 10% FBS; GIBCO, ThermoFisher Scientific) and 250′000 cells/well were cultured overnight in 250 ul of medium. Non-adherent cells were washed away and adherent peritoneal cells were treated for 16 h with or without LPS at a concentration of 100 ng/mL (InvivoGen, tlrl-smlps, LPS from S. minnesota R595). If indicated ATP (InvivoGen, tlrl-atp) was added at a concentration of 5 mM for 60 min prior to collection of culture supernatants. IL-1beta concentrations in cell culture supernatants were measured using MSD mouse IL-1beta assay kit (K152QPD; Meso Scale Discovery) according to the “alternative protocol 2” of the manufacturer's instructions (LLOD = 0.11 pg/mL).
Tlrl atp
Manufactured by InvivoGen
Tlrl-atp is a synthetic ligand that activates the ATP receptor P2X7. It is used in research applications to study the role of the P2X7 receptor in cellular signaling and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Tlrl 3pelps, by InvivoGen (2 mentions)
Tlrl msu, by InvivoGen (2 mentions)
Lpssm, by US Biological (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by US Biological (2 mentions)
Tlrl smlps, by InvivoGen (1 mentions)
70 um cell strainer, by Corning (1 mentions)
Tlrl nig, by InvivoGen (1 mentions)
Lipofectamine, by InvivoGen (1 mentions)
Tlrl mdpc, by InvivoGen (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
5 protocols using tlrl atp
1
Peritoneal Macrophage Isolation and Activation
2
Inflammasome Activation in Macrophages
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Primary peritoneal macrophages from C57BL/6 mice or THP1 cells were seeded in 24-well (5 × 105) or 6-well (2 × 106) culture plates. The next day, we removed medium and primed macrophages with LPS (InvivoGen tlrl-3pelps,100 ng/ml) for 3 h followed by 1,2-diol for 1 h. At the last, we treated macrophages with stimulation as follows: ATP (InvivoGen tlrl-atp, 5 mmol/L) or nigericin (InvivoGen tlrl-nig, 10 μmol/L) for 1 h and MSU (InvivoGen tlrl-msu, 200μg/ml) or SiO2 (InvivoGen tlrl-sio, 20 μg/ml) for 6 h. THP-1 cells should be treated with 100 ng/ml PMA for 12 h before seeding in culture plates.
3
IL-1β Secretion in Bone Marrow-Derived Cells
4
IL-1β Secretion in Bone Marrow-Derived Cells
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The BMDCs purified from wild type or GCN2−/− mouse tibia cultures were plated in 96 well microculture plates (1×105 cells/well) and primed for 8h with 100ng/ml LPS (InvivoGen, LPSSM) in RPMI 1640 (US Biological; R8999-04A) plus 1% dialyzed FBS (Gibco 26400-036). Cultures were further stimulated for 1hr with ATP (5mM) (InvivoGen, tlrl-atp). Following the stimulation, cell supernatants were collected and assayed for IL-1β by ELISA or Western blot. Cell pellets were lysed and assayed for the presence of pro-IL-1β and pro-Caspase 1.
5
THP-1 Macrophage Activation Protocol
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For experiments, THP-1 cells were seeded at a density of 1 × 106 cells/ml and differentiated into macrophages using 100 μg/ml PMA (Sigma-Aldrich, P1585) for 48 h. Medium was changed to serum-free medium 6 h before transfection with MDP (100 ng/ml; InvivoGen, tlrl-mdpc), or to serum-free medium containing ultrapure lipopolysaccharide (upLPS, 100 ng/ml; InvivoGen, tlrl-3pelps) 16 h before treatment with MSU (150 μg/ml; InvivoGen, tlrl-msu) or ATP (2 mM; InvivoGen tlrl-atp). For transfection, MDP (InvivoGen, tlrl-mdpc) was dissolved in DMSO and mixed with Lipofectamine® (InvivoGen, L3000–001) for 15 min before applying to the cell culture media. As a control, DSMO mixed with Lipofectamine® was used. MSU and ATP were applied onto the cells as suspensions. Likewise, BMDCs were collected at d 7 of BM cultures in differentiation medium, and seeded in new plates (0.5 × 106 cells/well in 12-well plates) in RPMI supplemented with 10% FCS. After 6 h, medium was changed for serum-free medium 6 h before transfection with MDP or treatment with MSU or ATP as described above.
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