In order to assess cell mobility, scratch assays were performed. BMDMs were seeded at 5.5 × 104 cells/well in IncuCyte® ImageLock 96-well plates in DMEM supplemented with 10% FBS (Sigma), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate Sigma, 22 μM β-mercaptoethanol (Gibco, Paisley, United Kingdom), and 10,000 I.U./mL PenStrep (Sigma), and rested overnight. The following day, the wells were scratched using the IncuCyte® WoundMaker, and the cells were immediately incubated with PBS, LPS-EK/IFN-γ, and IL-4/IL-10/TGF-β, frHMGB1, or dsHMGB1 at the indicated concentrations of 0.03–5 μg/mL (1.2–200 nM) in DMEM, supplemented with 1% FBS and L-glutamine and sodium pyruvate as described above. When using the TLR4 inhibitor CLI095 (Sigma), cells were pre-incubated with 5 μM CLI095 or with complete media containing 1% FBS for 1 h before the scratches were made. The plates were placed into the Incucyte® live cell analysis system and imaged every 2 h for 48 h. At each time point, the images and relative wound density were analyzed using the Incucyte® ZOOM software (2018A, Cell Migration Analysis Software Module, Essen Bioscience, Ann Arbor, MI, USA).
Cli095
Manufactured by Merck Group
Sourced in Spain, United States
CLI095 is a laboratory research product manufactured by Merck Group. It is a chemical compound used for scientific applications. The core function of CLI095 is to inhibit the activity of a specific cellular signaling pathway. No further details on the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Sb203580, by Merck Group (1 mentions)
P100 plates, by Corning (1 mentions)
Sp600125, by Merck Group (1 mentions)
β cyclodextrin, by Merck Group (1 mentions)
Sp600125, by StressMarq Biosciences (1 mentions)
A549 ccl 185, by American Type Culture Collection (1 mentions)
Cli 095, by Thermo Fisher Scientific (1 mentions)
H1299 crl 5803, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Antibiotic solution, by Thermo Fisher Scientific (1 mentions)
Skim milk powder, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Complete inhibitor cocktail, by Roche (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Cell Signaling Technology (1 mentions)
Ficoll paque, by Merck Group (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
6 protocols using cli095
1
Scratch Assay for Cell Mobility Assessment
2
7KCh Cytotoxicity Pathway Inhibition
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Cells were seeded in 12-well plates for cell viability or in P100 plates (Corning, New York, NY, USA) for RNA and protein purification at a density of 100.000 cells/well or 1.2 × 106 cells/plate, respectively. Then, cells were allowed to attach and reach 100% confluency for 24 h before removing the medium and replacing it with serum-free medium for 24 h. Treatments were then added and maintained for 24 h, with the exception of ELISAs that were maintained for 48 h, and some protein extracts that were collected at shorter times. Cells were treated with 8–20 μM 7KCh (Sigma-Aldrich, Madrid, Spain) alone or with 10 μM SA (PPQF, University of Alcalá, Madrid, Spain), 10 μM of the TLR4 inhibitor CLI-095 (Invivogen Inc., San Diego, CA, USA), 10–50 μM of the JNK inhibitor SP600125 (StressMarq Biosciences Inc., Victoria, BC, Canada) or 5–40 μM of the p38 inhibitor SB203580 (Sigma-Aldrich, Madrid, Spain). 7KCh was prepared in β-cyclodextrin (Sigma-Aldrich, Madrid, Spain) as previously described [15 (link),20 (link)], CLI-095 was dissolved in culture medium and SA, SP600125 and SB203580 were dissolved in DMSO (Sigma-Aldrich, Madrid, Spain).
3
Lung Adenocarcinoma Tissue Collection and Cell Line Cultivation
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Eighty-three cases of lung adenocarcinoma tissues and matched non-cancerous tissues were collected from patients who accepted surgery at the authors’ institution under the approval of Ethic Committee of the institution. These specimens were sent for routine pathological evaluation or instantly frozen in LN (liquid nitrogen).
Two human lung cancer cell lines, A549 (CCL-185™) and H1299 (CRL-5803™) were purchased from ATCC (Manassas, VA, USA) and cultured in RPMI 1640 with 10% FBS (Gibco®, Waltham, MA, USA). For LPS and CLI-095 treatment, 20 ng/ml LPS (Sigma, St. Louis, MI, USA) or 50 nmol/L CLI-095 (Invitrogen, Waltham, MA, USA) was used to stimulate the target cells for 4 h, then cells were harvested for further experiments.
Two human lung cancer cell lines, A549 (CCL-185™) and H1299 (CRL-5803™) were purchased from ATCC (Manassas, VA, USA) and cultured in RPMI 1640 with 10% FBS (Gibco®, Waltham, MA, USA). For LPS and CLI-095 treatment, 20 ng/ml LPS (Sigma, St. Louis, MI, USA) or 50 nmol/L CLI-095 (Invitrogen, Waltham, MA, USA) was used to stimulate the target cells for 4 h, then cells were harvested for further experiments.
4
Macrophage Polarization Assay
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RPMI 1640 was purchased from Lonza (Basel, Switzerland), and antibiotic solution (100 U/ml penicillin and 100 μg/ml streptomycin) was from Invitrogen Inc. (Carlsbad, CA, US). Monoclonal anti-human IκB-α, phospho-p38 mitogen-activated protein kinase (MAPK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were from Cell Signalling (Danvers, MA, US). Horseradish peroxidase-conjugated secondary antibodies were from Vector (Peterborough, UK). Anti-CCR2 mAb, anti-CD163 mAb, anti-IL-1β mAb, brefeldin, Fix, and Perm buffer solutions were from eBioscience/Affymetrix (Santa Clara, CA, US); anti-CD80 mAb and anti-CD206 mAb were from BD Biosciences Pharmigen (San Diego, CA, US). The cOmplete™ inhibitor cocktail was from Roche Diagnostics (Mannheim, Germany). Fetal bovine serum (FBS), Ficoll-Paque (density 1.077 ± 0.001), Percoll, dexamethasone, curcumin, CLI-095, skim milk powder as well as other analytic grade chemical agents were from Sigma-Aldrich. Ultrapure LPS (LPS-EB), zymosan (ZYMO), Pam3CSK4, and Poly(I:C) were from InvivoGen (San Diego, CA, USA). IL-4, IL-13, and colony stimulating factor-1 (CSF-1) were from ImmunoTools (Friesoythe, Germany). The curcumin analogues GG6 and GG9 were synthesized as described elsewhere [13 (link)].
5
Fatty Acid Stimulation of Macrophages
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Palmitic acid (Cat. No. P0500, Sigma, Sigma chemicals, St Louis, MO, USA), Oleic acid (Cat. No. O1008, Sigma, Sigma chemicals), Elaidic acid (Cat. No. E4637, Sigma, Sigma chemicals), LPS (Cat. No. L8274, Sigma, Sigma chemicals), CLI095 (intracellular TLR4 antagonist; Cat. No. tlrl-cli95, InvivoGen, San Diego, CA, USA), fatty acid free (FAF) BSA, FAF-BSA; Cat. No. 10775835001, Roche, Mannheim, Germany) and BSA (Cat. No. A7888 Sigma, Sigma chemicals).
6
Phagocytic Internalization of Salmonella
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To detect bacterial phagocytosis, monocyte-macrophages were infected with S. typhimurium. Before infection, monocyte-macrophages were washed three times in PBS without antibiotics and then mixed with S. typhimurium at an MOI of 10 or 100, centrifuged at 100 × g for 10 min, and incubated at 37°C in a 5% CO 2 incubator for 5, 15, or 30 min. Cells were then washed twice with PBS and incubated in RPMI1640 medium containing 10% FBS and gentamicin (30 μg/mL) for 30 min to kill any non-internalized bacteria. To monitor the phagocytized bacteria at different time points, cells were washed three times with PBS and then lysed with 1% Triton X-100. After serial dilution, the last dilution was spread onto agar plates. After overnight culture of the bacteria, the number of viable CFUs was recorded to estimate the number of internalized bacteria.
To determine whether TLR4-PI3K signaling participates in bacterial internalization, we used the TLR4 and PI3K inhibitors CLI095 and LY294002 (Sigma-Aldrich) prior to infection with S. typhimurium to inhibit their respective signaling. Bacterial internalization-associated genes involved in these signaling pathways were evaluated by qRT-PCR as described above. Primer sequences are shown in Table 2.
To determine whether TLR4-PI3K signaling participates in bacterial internalization, we used the TLR4 and PI3K inhibitors CLI095 and LY294002 (Sigma-Aldrich) prior to infection with S. typhimurium to inhibit their respective signaling. Bacterial internalization-associated genes involved in these signaling pathways were evaluated by qRT-PCR as described above. Primer sequences are shown in Table 2.
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