Flagellin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Flagellin is a protein that is a major component of the flagella in many bacteria. It serves as the structural subunit that assembles to form the flagellum, which is responsible for the motility of the bacterial cell.

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7 protocols using flagellin

NLRP3, NLRC4, and AIM2 Inflammasome Activation

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To trigger conventional NLRP3 inflammasome activation, J774A.1 cells were primed with 0.25 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) for 3 h and then treated with 2.5 mM ATP (Sigma-Aldrich) for 30 min with or without 10 µM Y-VAD (InvitroGen, Carlsbad, CA, USA), a selective caspase-1 inhibitor. To induce NLRC4 or AIM2 inflammasome, J774A.1 cells were primed as described above and then transfected with 0.5 μg/ml flagellin (InvitroGen) or 1 μg/ml poly(dA:dT) (InvitroGen) for 3 h, using Lipofectamine 2000 (InvitroGen) according to the manufacturer's protocol.
Flag-tagged caspase-11 (Flag-Caspase-11) and either Flag-tagged wild-type GSDMD (Flag-GSDMD [WT]) or D276A mutant GSDMD (Flag-GSDMD [D276A]) were transfected into HEK293T cells. Flag-GSDMD (D276A) was constructed in our laboratory. Then, HEK293T cells were treated with 20 μg/ml LPS for 4 h with or without 10 µM Z-VAD (InvitroGen), a pan-caspase inhibitor. Alternatively, HEK293T cells were transfected with Flag-tagged wild-type C-terminal domain of GSDMD (Flag-GSDMD-CT [WT]), wild-type N-terminal domain of GSDMD (Flag-GSDMD-NT [WT]), or 4A mutant N-terminal domain of GSDMD (Flag-GSDMD-NT [4A]) and cultured for 48 h.

Park C.H., Lee H.S., Kwak M.S, & Shin J.S. (2021). Inflammasome-Dependent Peroxiredoxin 2 Secretion Induces the Classical Complement Pathway Activation. Immune Network, 21(5), e36.

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Immunoblotting Analysis of Recombinant Proteins

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For immunoblotting analysis, recombinant proteins resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto nitrocellulose membrane with a mini Trans-Blot transfer electrophoresis unit (Bio-Rad). The membranes were blocked for 2 h at RT in TBST blocking buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk powder to prevent nonspecific binding and then incubated with specific polyclonal primary antibodies raised against Cap protein, flagellin (Invitrogen), β-actin (Santa Cruz), or monoclonal antibody against histidine tag (Invitrogen) at RT for 2 h. The membranes were washed three times with TBST buffer and incubated for 2 h at RT with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in blocking buffer. Immunoreactive bands were visualized by enhanced chemiluminescence system (Kodak Image Station 4000R).

Zhang C., Zhu S., Wei L., Yan X., Wang J., Quan R., She R., Hu F, & Liu J. (2015). Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice. PLoS ONE, 10(6), e0129617.

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TLR Reporter Cell Line Assay

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A TLR reporter cell line system that monitored TLR activity using secreted embryonic alkaline phosphatase (SEAP) induced by NF-κB and AP-1 activation was used. HEK-Blue™ Detection (InvivoGen) reacts with SEAP to turn the medium color purple/blue, and the color intensity can be measured with a spectrophotometer. The TLR reporter cell lines (InvivoGen) were cultured in DMEM containing 10% FBS and 1× antibiotics. When the cells reached 80% confluent, they were washed with PBS, and detection medium was added to harvest the cells using a scraper. Cells (5 × 10⁴ cells/well; 180 μl) were seeded in a 96-well plate, and 20 μl of PBS, samples, PAM3 (Invitrogen, Waltham, Massachusetts, USA), LPS (Invitrogen), poly I:C (Invitrogen), flagellin (Invitrogen), R848 (Invitrogen), or CpG-ODN (Invitrogen) was added to each well. Then, the cells were cultured at 37 °C, 5% CO₂, and 90% humidity for 20 h, and the absorbance at 630 nm was recorded.

Kim C.U., Jeong Y.J., Lee P., Lee M.S., Park J.H., Kim Y.S, & Kim D.J. (2022). Extracellular nucleoprotein exacerbates influenza virus pathogenesis by activating Toll-like receptor 4 and the NLRP3 inflammasome. Cellular and Molecular Immunology, 19(6), 715-725.

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Adoptive Transfer of CD45.1 Myeloid Cells

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MOPs from pooled BM of CD45.1 female donor mice (n = 5) were flow cytometrically sorted and transferred intravenously (i.v.) via the retro-orbital plexus into age-matched female CD45.2 recipient mice (n = 8) at 100,000 cells in 200 μl PBS per mouse. At 24 h after adoptive transfer, recipient mice were anesthetized by methoxyflurane inhalation, and intranasally (i.n.) inoculated with 1 μg Flagellin (Invitrogen) in 50 μl PBS per mouse (n = 4) or with 50 μl PBS (n = 4).

Lei X., Palomero J., de Rink I., de Wit T., van Baalen M., Xiao Y, & Borst J. (2021). Flagellin/TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages. Frontiers in Immunology, 12, 621665.

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Metabolite-modulated PGE2 secretion in cMSCs

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In stimulation experiments, cMSCs were plated in 96-well plates at 20,000 cells/well in low glucose DMEM (containing 10mM HEPES, penicillin-streptomycin, 10% FBS) and allowed to adhere overnight. The following morning, cells were fed fresh media, stimulated with ligands and incubated at 37°C for 24 hours. For TLR screen, cMSCs were individually stimulated with Pam3CSK4, LPS, flagellin, Pam2CSK4, poly IC (Invitrogen) at indicated doses or vehicle (endotoxin free water, Invitrogen). For the metabolite screen, cMSCs were stimulated with 100ng/ml of Pam3CSK4 in the presence or absence of bacterial metabolites (100 µM). For secondary validation, metabolites at 100µM, 10µM and 1µM were used. For receptor agonists, cMSCs were stimulated with 100ng/ml Pam3CSK4 in the presence or absence of GW4064 (Tocris), INT747 (MedChem Express) or INT-777 (MedChem Express). 24-hour culture supernatants were collected from the treatments and PGE2 concentration was assayed. In some experiments, cells were washed with PBS and lysed in RIPA (Sigma) buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific). These lysates were then assayed for PGE2. All the experiments utilized cMSCs between passage number 5 and 8.

Jain U., Lai C.W., Xiong S., Goodwin V.M., Lu Q., Muegge B.D., Christophi G.P., VanDussen K.L., Cummings B.P., Young E., Hambor J, & Stappenbeck T.S. (2018). Temporal regulation of the bacterial metabolite deoxycholate during colonic repair is critical for crypt regeneration. Cell host & microbe, 24(3), 353-363.e5.

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Keratinocyte Culture and Stimulation Protocol

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Normal human epidermal keratinocytes (NHEKs) were purchased from MilliporeSigma and cultured for 3–4 passages using the EpiGRO™ Human Epidermal Keratinocyte Complete Media Kit (MilliporeSigma, Burlington, MA, USA). Human skin-equivalent models (HSEMs) were purchased from MetTek. HSEMs were cultivated according to the manufacturer’s instructions. NHEKs were grown in a humidified atmosphere containing 5% CO₂ and 95% air atmosphere. Cells were seeded at a density of 2 × 10⁴ cells/well in a flat-bottomed 96-well microculture plate and were cultured until they reached 90% confluence, then the media was changed to fresh medium without hydrocortisone. After further cultivation for 24 h, keratinocytes were stimulated using previously described methods with CCs [52 (link),53 (link)] that included: TNF-α (20 ng/mL, R&D system), IL-4 (100 ng/mL, R&D system), IL-13 (100 ng/mL, R&D system) and flagellin (100 ng/mL; Peprotech, Inc., Seoul, Korea). Kushenol F pretreatment was performed for 1 h before keratinocyte activation.

Jo S., Gong E.Y., Yoo W., Choi H., Jung D., Noh K.H., Kim S., Kim S.H, & Lee H.K. (2022). Anti-Itching and Anti-Inflammatory Effects of Kushenol F via the Inhibition of TSLP Production. Pharmaceuticals, 15(11), 1347.

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Inflammatory Response in Caco-2 Cells

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Bacteria were added in the apical compartment of the Caco-2 cells inserts. Basolateral compartment of the inserts were filled with DMEM without FCS or antibiotics. Inserts were then incubated for 16 h at 37°C in a 5% CO₂ incubator when pro-inflammatory signals were applied. Deoxynivalenol (DON, 10 μM final concentration, obtained from Romer lab) was added to the apical compartment, while human recombinant IL-1β (20 ng/ml final concentration, obtained from Peprotech), or flagellin (1 μg/ml final concentration, extracted from Salmonella typhimurium, obtained from Invivogen) were added basolaterally to Caco-2. Epigallocatechin gallate (EGCG, 10 μM final concentration, obtained from Sigma Aldrich) was added to the apical compartment and used as positive anti-inflammatory control. After 6 h at 37°C, basolateral media were collected and stored at −80°C before IL-8 cytokine measurement by ELISA (BD Biosciences) was performed. TEER was measured using a Volt/Ohm meter (Millipore).

Rhayat L., Maresca M., Nicoletti C., Perrier J., Brinch K.S., Christian S., Devillard E, & Eckhardt E. (2019). Effect of Bacillus subtilis Strains on Intestinal Barrier Function and Inflammatory Response. Frontiers in Immunology, 10, 564.

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