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Lonomycin

Manufactured by Merck Group
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Lonomycin is a polyether ionophore compound produced by the bacterium Streptomyces longsporoflavus. It functions as a potassium ionophore, capable of facilitating the transport of potassium ions across cell membranes.

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Lab products found in correlation

Sigma blend collagenase type f, by Merck Group (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Brefeldin a, by Merck Group (2 mentions) Mouse il 17a elisa duo set kit, by R&D Systems (1 mentions) Golgistop, by BD (1 mentions)

5 protocols using lonomycin

1

Isolation and Activation of Lamina Propria Mononuclear Cells

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Samples of the total small intestine were rinsed in PBS and then treated with 2 mmol/L EDTA in PBS for 30 min to remove the epithelial cells. The residue was then digested with Sigma Blend Collagenase Type F (Sigma-Aldrich Co, Ltd., St Louis, MO) for 20 min and separated in the Percoll gradient to obtain LPMCs. In the primary cultures, 1 × 106 cells of LPMCs per well were cultured in 100 μL of 10% FCS/RPMI1640 for 24 h. At that time, 40 µL each of PMA (Phorbol 12-Myristate 13 Acetate) (Merck KGaA, Darmstadt, Germany) and lonomycin (Merck KGaA) were added to the culture medium. After culturing for 24 h, the supernatant was collected and stored in a freezer at −80 °C until it was used for ELISA. LPMCs were washed with PBS and stored in a freezer at −80 °C until they were used for mRNA expression analyses, ELISA, and flow cytometry.
Kawashima R., Tamaki S., Hara Y., Maekawa T., Kawakami F, & Ichikawa T. (2023). Interleukin-13 Mediates Non-Steroidal Anti-Inflammatory-Drug-Induced Small Intestinal Mucosal Injury with Ulceration. International Journal of Molecular Sciences, 24(19), 14971.
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2

Isolation and Activation of Lamina Propria Mononuclear Cells

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Samples of the total small intestine were rinsed in PBS and then treated with 2 mmol/L EDTA in PBS for 30 min to remove the epithelial cells. The residue was then digested with Sigma Blend Collagenase Type F (Sigma-Aldrich Co, Ltd., St Louis, MO) for 20 min and separated in the Percoll gradient to obtain LPMCs. In the primary cultures, 1 × 106 cells of LPMCs per well were cultured in 100 μL of 10% FCS/RPMI1640 for 24 h. At that time, 40 µL each of PMA (Phorbol 12-Myristate 13 Acetate) (Merck KGaA, Darmstadt, Germany) and lonomycin (Merck KGaA) were added to the culture medium. After culturing for 24 h, the supernatant was collected and stored in a freezer at −80 °C until it was used for ELISA. LPMCs were washed with PBS and stored in a freezer at −80 °C until they were used for mRNA expression analyses, ELISA, and flow cytometry.
Kawashima R., Tamaki S., Hara Y., Maekawa T., Kawakami F, & Ichikawa T. (2023). Interleukin-13 Mediates Non-Steroidal Anti-Inflammatory-Drug-Induced Small Intestinal Mucosal Injury with Ulceration. International Journal of Molecular Sciences, 24(19), 14971.
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3

Flow Cytometric Analysis of Splenic Cells

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Spleens from mice in each group were removed to remove surrounding excess tissue, which was triturated, digested, filtered through a 70 mm mesh, and centrifuged to obtain a single‐cell suspension. Phorbol 12‐myristate 13‐acetate, brefeldin A, and lonomycin (Sigma‐Aldrich) were added to the cells, and incubation was continued at 37°C in 5% CO2. The fluorescein‐labeled monoclonal antibody CD4‐FITC (Cell Signaling Technology) was then added to incubate in the dark at room temperature. After treatment with the fixative for ruptured nuclear membrane, the intracellular antibody FOXP3‐APC was added. Finally, data were acquired with detection in flow cytometry.
Wang A., Ma X., Wei F., Li Y., Liu Q, & Zhang H. (2023). Evidence on the therapeutic role of thiolutin in imiquimod‐induced psoriasis‐like skin inflammation in mice. Immunity, Inflammation and Disease, 11(7), e877.
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4

Measuring IL-17A in Immune Cell Assays

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Cells derived from the nasal mucosa, spleen, or colon were stimulated for 4–5 hours with PMA (50 ng/ml), lonomycin (500 μg/ml) (Sigma Aldrich), and golgi stop (BD) (1 μg/ml), or stimulated overnight with WCA (10 μg/ml) followed by Golgistop (1 μg/ml) for 4–5 hours. Following these incubations, cells were harvested and prepared for surface and intracellular cytokine staining as described above. Nasal cell suspensions were aliquoted into 24-well TC plates in complete tissue culture media (TCM) and stimulated with WCA (10 μg/ml ) for 7 days. Supernatants were harvested and IL-17A was detected in the supernatants by ELISA as per the mouse IL-17A ELISA DUO set kit instructions (reference DY421; R&D Systems). IL-17A was detected in samples of WCA-stimulated whole blood as previously described18 . A value of 8 pg/ml was set as the lower limit of detection of IL-17A cytokine protein by ELISA and is represented by the dotted line in IL-17A cytokine detection graphs.
O’Hara J.M., Redhu N.S., Cheung E., Robertson N.G., Patik I., Sayed S.E., Thompson C.M., Herd M., Lucas K.B., Conaway E., Morton C.C., Farber D.L., Malley R, & Horwitz B.H. (2019). Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization. Mucosal Immunology, 13(1), 172-182.
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5

Th1/Th2 Cell Ratio Analysis

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Levels of IFN‐γ‐producing Th cells (Th1 cells; CD4+ T lymphocytes with IFN‐γ without IL‐4) and IL‐4‐producing Th cells (Th2 cells; CD4+ T lymphocytes with IL‐4 without IFN‐γ) were analyzed to evaluate the Th1/Th2 ratio by SRL Laboratory, Tokyo, Japan. Laser flow cytometry (Fascinator II; BD Biosciences) was used to measure the levels of Th cells with phorbol 12‐myristate 13‐acetate, lonomycin, Brefeldin A (Sigma‐Aldrich Corp.), CD4 R‐phycoerythrin‐cyanine (PC)‐5 (Immunotec), and fluorescein isothiocyanate (FITC)/IL‐4‐PE (BD Biosciences). After the activated whole blood samples were stained with anti‐CD4‐PC5‐conjugated monoclonal antibodies, lysis of red blood cells and specific intracellular staining were performed according to the manufacturer's instruction using Fast ImmuneTM IFN‐FITC/IL‐4‐PE (Becton Dickinson Biosciences). The Th1/Th2 cell ratio was calculated using the ratio of IFN‐γ‐positive to IL‐4‐positive cells.
Ichikawa T., Negishi Y., Kasano S., Yokote R., Yonezawa M., Ouchi N., Kuwabara Y., Suzuki S, & Takeshita T. (2022). Upregulated serum granulysin levels in women with antiphospholipid antibody‐associated recurrent miscarriage are downregulated by heparin treatment. Reproductive Medicine and Biology, 21(1), e12460.
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