A spontaneously transformed type II mouse lung alveolar epithelial cell line [19] (link) (C10) was cultured as described previously [20] (link). A C10 cell line stably expressing NF-κB luciferase was generated as previously described [21] (link). Briefly, C10 cells were transfected with the plasmid, 6 κB-tk-luc, in the presence of the plasmid PSV-2Neo (Promega), conferring a resistance to neomycin. Following incubation with the antibiotic, geneticin, antibiotic-resistant colonies were propagated. C10 cells were starved in medium containing 0.5% FBS for 16 h prior to exposure of 1 µg/mL LPS (List Biological Laboratories, Inc).
Lipopolysaccharides (lps)
Manufactured by List Biological Laboratories
Sourced in United States
LPS is a laboratory product manufactured by List Biological Laboratories. It is a purified lipopolysaccharide extracted from gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Poly 1 c, by InvivoGen (5 mentions)
Il 1β, by R&D Systems (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Ifn γ, by R&D Systems (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Fujifilm (1 mentions)
Lipopolysaccharides (lps), by Fujifilm (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Elisa kit, by Thermo Fisher Scientific (1 mentions)
Vetscan hm5 hematology analyzer, by Abaxis (1 mentions)
Pha568487, by Bio-Techne (1 mentions)
C57bl 6jrj mice, by Janvier Labs (1 mentions)
8 br camp, by Merck Group (1 mentions)
Elisa, by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
L glu, by Thermo Fisher Scientific (1 mentions)
Srp3093, by Merck Group (1 mentions)
213 ilb 005, by R&D Systems (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
23 protocols using lipopolysaccharides (lps)
1
Generation of NF-κB Luciferase-Expressing Alveolar Cell Line
2
Silica-induced IL-1β Secretion in BMDMs
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BMDMs (1×105/well) were seeded into 48-well plates and cultured overnight. Then cells were primed with 10 ng/mL lipopolysaccharide (LPS; List Biological Laboratories, Campbell, CA). Five hours later, cells were stimulated with the indicated dose and size of silica particles or with 1 mM ATP (Wako, Osaka, Japan) for four hours at 37°C. For some experiments, LPS-primed BMDMs were pretreated with 20 μM of cytochalasin D (Cyto D; Wako, Japan), or 200 nM of Bafilomycin A1 (Sigma-Aldrich, St Louis, MO) for 1 hour, and were then stimulated with silica particles for 4 hours. The amount of IL-1β in cell culture supernatants was measured by an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA) according to the manufacturer’s instructions.
3
Synovial Fibroblast Stimulation Assay
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Synovial tissues were obtained from patients with RA diagnosed according to the 1987 American College of Rheumatology criteria for classification of RA,[19 (link)] who underwent joint reconstruction surgery (n = 32). Synovial tissues from patients with osteoarthritis (OA, degenerative disease) served as controls (n = 27). The study was approved by the Kantonale Ethikkommission der Kanton Zurich, with the approval numbers 475 and 515. All patients provided a written informed consent and the study was approved by the local Ethics committee. SF cultures were established by dispase digestion of synovial tissue as described elsewhere.[20 ] Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. SF of passages 4–8 were used for all experiments. RASF and OASF were stimulated or not for 24 hours with Poly(I:C) (10ug/ml; Invivogen), LPS (100ng/ml; List Biological Laboratories) or TNFα (10 or 100ng/ml; R&D) alone or in combination with IL1β (1ng/ml; R&D).
4
Evaluating PHA 568487 Immunomodulatory Effects
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To evaluate the possible immunomodulatory effects of PHA 568487 in vivo, the air pouch model of acute inflammation was used [12 (link),25 (link)]. At 8 weeks of age, male C57BL/6JRj mice (Janvier Labs, Le Genest-Saint-Isle, France) were injected with 3 mL of sterile air between the scapulae under isoflurane anesthesia. Three days later, the air pouch was re-filled with 3 mL of sterile air. On the sixth day, the mice received an intraperitoneal injection of either PBS (Thermo Fisher Scientific, Waltham, MA, USA) or PHA 568487 (Tocris Bioscience, Ellisville, MO, USA) at 12.4 μmol kg−1 (5 mg/kg) or 123.4 μmol kg−1 (50 mg/kg). Fifteen minutes later, all mice were injected with LPS (10 μg, List Biological Laboratories Inc., Campbell, CA, USA) directly into the pouch. The mice were sacrificed six hours after the LPS injection by an overdose of pentobarbital (Apoteket AB, Stockholm, Sweden). Finally, the pouch cavity was flushed with 2 mL of PBS with 1.6 mmol L−1 of EDTA, and the exudate was collected. The white blood cell count of the exudate was analyzed using the Vetscan HM5 Hematology Analyzer (Abaxis, Union City, CA, USA) and thereafter centrifuged for 5 min at 400× g. Supernatants were stored at −80 °C until multiplex analysis.
5
Macrophage Infection and Cytokine Response
6
Postnatal Neuroinflammation in Mice
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All animals were kept on a 12 h dark-light cycle and standard housing conditions with ad libitum food access. On postnatal day 5 (P5), C57Bl/6J mice were randomly assigned to 4 experimental groups: LPS (1 mg/kg, #423, List Biological Laboratories, Campbell, CA, USA)-injected mice (n = 12, ♂ = 6, ♀ = 6) and saline (1 mg/kg, Sigma Aldrich, St. Louis, MO, USA)-injected mice (n = 12, ♂ = 6, ♀ = 6). Injections were performed intraperitoneally. This age in mice represents a critical brain developmental stage equivalent to preterm human infants [14 (link)]. Morphological analysis was performed 7 days after injection (P12). The P12 time point was studied as it corresponds to a stage in mouse brain development with high rate of brain growth, including gliogenesis, increasing axonal and dendritic density, and with significant brain plasticity activity [14 (link)]. Animals were anesthetized deeply with an intraperitoneal injection of pentobarbital (20 μL per animal, 60 mg/mL), followed by transcardial perfusion with histofix (Histolab products AB, Vastra Frölunda, Sweden) and collection of brains. One brain hemisphere (left or right) was randomly selected for each animal to have equal number of each hemisphere per group, immersed in the same fixative solution, and stored at 4°C for further brain structural quantification.
7
Isolation and Culture of Bone Marrow-Derived Dendritic Cells
8
U937 Macrophage Polarization Protocol
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U937 cells were seeded in TomoDishes at about per dish with complete media and PMA (Sigma-Aldrich 79346, ). The complete media consisted of RPMI-1640 (Gibco, 11875-093), 10% heat-inactivated FBS (Corning, 35-010-CV), 1% P/S, 1%L-Glu (Gibco, 200 mM, 25030081), and 1% HEPES (Gibco, 1M, 15630-080). The seeding day is counted as day 0.
For M1 polarized cells, on day 3, PMA media is removed, and cells are washed with PBS. Thereafter, polarizing media consisting of complete media with LPS (List Biological Laboratories, 421, ) and IFN-y (Sigma-Aldrich, SRP3093, ) were added to the dish. The cells were incubated for another 2 days. On day 5, the media was removed, and cells were washed with PBS before fixing.
For M2 polarized cells, on day 3, PMA media is removed, and cells are washed with PBS. Thereafter, polarizing media consisting of complete media with IL-4 (Sigma-Aldrich, SRP3093, ) and IL-13 (R&D Systems, 213-ILB-005, ) were added to the dish. The cells were incubated for another 2 days. On day 5, the media was removed, and cells were washed with PBS before fixing.
The cells were fixed with 4% PFA at room temperature for 15 min. It was then washed with PBS two times. PBS was once again added to fully submerge the fixed cells before imaging.
For M1 polarized cells, on day 3, PMA media is removed, and cells are washed with PBS. Thereafter, polarizing media consisting of complete media with LPS (List Biological Laboratories, 421, ) and IFN-y (Sigma-Aldrich, SRP3093, ) were added to the dish. The cells were incubated for another 2 days. On day 5, the media was removed, and cells were washed with PBS before fixing.
For M2 polarized cells, on day 3, PMA media is removed, and cells are washed with PBS. Thereafter, polarizing media consisting of complete media with IL-4 (Sigma-Aldrich, SRP3093, ) and IL-13 (R&D Systems, 213-ILB-005, ) were added to the dish. The cells were incubated for another 2 days. On day 5, the media was removed, and cells were washed with PBS before fixing.
The cells were fixed with 4% PFA at room temperature for 15 min. It was then washed with PBS two times. PBS was once again added to fully submerge the fixed cells before imaging.
9
RAGE Knockdown Modulates TNF-α and LPS Responses
10
Infection and Stimulation of MDDCs
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