Flagellin fla st

Manufactured by InvivoGen

Flagellin (FLA-ST) is a protein derived from Salmonella Typhimurium that functions as a Toll-like receptor 5 (TLR5) agonist. It can be used for cell stimulation and immune response studies.

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Salmonella typhimurium flagellin (FLA-ST) Flagellin (FLA-ST ultrapure) Flagellin-ST FLA-ST Flagellin

4 protocols using flagellin fla st

Breast Cancer Cell Lines Protocol

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Human breast epithelial cell line MCF-10A, human breast cancer cell lines MCF-7, MDA-MB-435s, MDA-MB-468, T47D, MDA-MB-231 and MDA-MB-431 were originally purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Antibodies against MyD88, cyclin D1 and p27Kip1 were purchased from purchased from Abcam. Antibody against MAP1S (4G1) was from Precision Antibody. Antibody against β-actin was purchased from Santa Cruz Biotechnology. Flagellin (FLA-ST) was purchased from InvivoGen. NF-κB and AP-1 luciferase reporter was described previously [20] (link).

Shi M., Yao Y., Han F., Li Y, & Li Y. (2014). MAP1S Controls Breast Cancer Cell TLR5 Signaling Pathway and Promotes TLR5 Signaling-based Tumor Suppression. PLoS ONE, 9(1), e86839.

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Stimulation of Immune Cells with TLR Ligands

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TLR ligands: Pam₃CSK4, PGN, LTA, Poly(I:C), CL075, CL097, LPS, Flagellin (FLA-ST), Flagellin (FLA-ST ultrapure grade), R848, CpG-A (ODN2216), CpG-B (ODN2006) and CpG-C (ODN M362) were purchased from Invivogen. Poly (G3) was purchased from Sigma. Mycobacterium tuberculosis (MT) killed bacteria and Bordetella pertussis (BP) inactivated bacteria were purchased from BD Diagnostic Systems. Yellow fever vaccine (YFVAX) was a generous gift from Dr. B. Pullendran, Emory Vaccine Center at Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USA. ELISA kits for GM-CSF, IL-2, and IL-12 were purchased from R&D systems. Fetal calf serum and human serum were purchased from Gemini. Carboxyfluorescein succinimidyl ester (CFSE) was purchased from Invitrogen. Mouse fibroblast L cell line (L-cell) and OX40 ligand expressing L cells (L-cell/OX40L) generated in our laboratory were maintained with RPMI cultured medium containing 10% fetal calf serum, 1% Glutamax, and 1% penicillin-streptomycin. FL-specific T-cell lines and clones generated from intratumoral T cells were described previously.^{29 (link)}

Voo K.S., Foglietta M., Percivalle E., Chu F., Nattamai D., Harline M., Lee S.T., Bover L., Lin H.Y., Baladandayuthapani V., Delgado D., Luong A., Davis R.E., Kwak L.W., Liu Y.J, & Neelapu S.S. (2014). Selective targeting of Toll-like receptors and OX40 inhibit regulatory T cell function in follicular lymphoma. International journal of cancer. Journal international du cancer, 135(12), 2834-2846.

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Transient Transfection and Luciferase Assay

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HEK 293T cells were seeded in a 96‐well plates at 2 × 10⁴ cells/well overnight, and cells were transiently transfected with FuGENE^® 6 (Promega) for 24 h for a total of 0.4 μg of DNA consisting of 50 ng TLR plasmids, 200 ng of pBIIXLuc reporter plasmid, 5 ng of control Renilla luciferase (pRL‐null, Promega) and 50 ng of myc‐PumA expression vector. The total amount of DNA was kept constant by adding empty vector. Where indicated, cells were treated with E. coli LPS (1 μg/ml) and Flagellin FLA‐ST (1 μg/ml), all obtained from InvivoGen, for 6 h. In the case of IL‐1β and TNFR, endogenous receptors were stimulated with IL‐1β (100 ng/ml) and TNFα (100 ng/ml), respectively. Cells were then lysed and luciferase activity measured using Dual‐Glo Luciferase Assay System (Promega).

Imbert P.R., Louche A., Luizet J., Grandjean T., Bigot S., Wood T.E., Gagné S., Blanco A., Wunderley L., Terradot L., Woodman P., Garvis S., Filloux A., Guery B, & Salcedo S.P. (2017). A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors. The EMBO Journal, 36(13), 1869-1887.

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TLR Ligand Stimulation of Differentiated Cells

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After differentiation and treatment with FS, the cells were stimulated with TLR ligands in complete media; Iscove’s media (Merck, Kenilworth, NJ) supplemented with 10% fetal bovine serum (Biological Industries, Beit HaEmek, Israel), 200 mmol/L L-glutamine (Sigma, St. Louis, MO), and 50 μg/mL gentamicin. For stimulation, complete media containing 10 ng/mL LPS (LPS-EK), 10 ug/mL PGN (PGN-BS), 50 ng/mL flagellin (FLA-ST), or 5 umol/L CpG oligonucleotides (ODN2006) was added (all Invivogen). After 24 hours of stimulation, the supernatants were collected and stored at -20°C. The cells were collected and used either for flow cytometry or for quantitative PCR analysis.

Maasfeh L., Härtlova A., Isaksson S., Sundin J., Mavroudis G., Savolainen O., Strid H., Öhman L, & Magnusson M.K. (2021). Impaired Luminal Control of Intestinal Macrophage Maturation in Patients With Ulcerative Colitis During Remission. Cellular and Molecular Gastroenterology and Hepatology, 12(4), 1415-1432.

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