Ionomcyin

Manufactured by Merck Group

Ionomycin is a laboratory reagent used in cell biology research. It is a calcium ionophore that facilitates the movement of calcium ions across cell membranes, leading to an increase in intracellular calcium levels. This property makes Ionomycin a useful tool for studying calcium-dependent cellular processes.

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3 protocols using ionomcyin

In vitro Cytokine and Proliferation Assay

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To determine cytokine production upon in vitro stimulation, splenocytes were stimulated with 20 ng/mL PMA (Sigma) and 0.5 μg/mL ionomcyin (Sigma), and incubated with 1 μL/mL Golgi Plug protein transport inhibitor (Becton Dickinson) for 4 h. Cells were stained as described above. To determine proliferation and IFNγ production upon in vitro TCR-mediated stimulation splenocytes were enriched for T cells using CD19 microbeads depletion (Miltenyi Biotec) or pan-T cell Isolation Kit II (Miltenyi Biotec) on LS columns (Miltenyi Biotec). Cells were plated in a 96-well plate coated with anti-CD3 (145-2C11) (BD) and anti-CD28 (37.51) (BD) was added to the medium.

Kwesi-Maliepaard E.M., Aslam M.A., Alemdehy M.F., van den Brand T., McLean C., Vlaming H., van Welsem T., Korthout T., Lancini C., Hendriks S., Ahrends T., van Dinther D., den Haan J.M., Borst J., de Wit E., van Leeuwen F, & Jacobs H. (2020). The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells. Proceedings of the National Academy of Sciences of the United States of America, 117(34), 20706-20716.

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Comprehensive Lung Immune Cell Profiling

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Lungs were harvested, digested, and lung single-cell suspensions were restimulated with 50 ng/ml of PMA (Sigma Aldrich), 1 μg/ml of ionomcyin (Sigma Aldrich) and 0.07% Golgi-Stop (BD Biosciences) for 5 hours at 37⁰C in RPMI+10% FBS. Following restimulation, cells were stained with viability dye (UV Ghost dye 450, Tonbo), blocked with an anti-FcR antibody (clone 2.4G2), and surface stained with biotin-labeled anti-CD3 (clone 17A2), PE-Cy5 anti-CD4 (clone 129.19), BV786 anti-CD90.2 (clone 53–2.1), Alexa Fluor 700 anti-CD45 (clone 30-F11), Alexa Fluor 488 anti-CD25 (clone OC61), PE-Cy7 anti-CD127 (clone A7R34) and FITC anti-γδTCR (clone GL3) antibodies, followed by APC-Cy7 streptavidin staining (1:250). Cells were then fixed, permeabolized using the Foxp3/transcription factor staining kit (Tonbo), and intracellularly stained with PE-IL-13 (clone eBio13A), PE-Cy7 anti-IL-17A (clone eBio17B7), and/or PE-IL-4 (clone 11B11). Flow cytometry analysis was conducted on LSRII flow cytometer, and all data were processed using FlowJo software version 10.

Fuseini H., Yung J.A., Cephus J.Y., Zhang J., Goleniewka K., Poloshukin V.V., Peebles RS J.r, & Newcomb D.C. (2018). Testosterone decreases house dust mite-induced type 2 and IL-17A mediated airway inflammation. Journal of immunology (Baltimore, Md. : 1950), 201(7), 1843-1854.

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Intracellular Cytokine Staining Assay

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Splenocytes were seeded into 96-well plates at a density of 2 x 10⁶ cells/well. Brefeldin A (Sigma, St. Louis, MO) and monensin (Sigma, St. Louis, MO) added to each well at final concentrations of 10 μg/ml. Env or Gag peptide pool was added to each well at a concentration of 2 μg/ml. Phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) and ionomcyin (Sigma, St. Louis, MO) at concentrations of 50 ng/ml and 500 ng/ml, respectively, were the positive controls. Cells were incubated for 6 hours at 37°C, 5% CO₂. Afterwards, cells were washed and incubated with anti-CD4 (clone: GK1.5; BD Biosciences, San José, CA) and anti-CD8a (clone: 53–6.7; BD Biosciences, San José, CA) cell membrane antibodies. After 45 minutes, cells were washed, fixed in BD Perm/Fix (BD Biosciences, San José, CA), permeabilized, and stained for intracellular cytokines IL-2 (clone: JES6-5H4; BD Biosciences, San José, CA), IL-4 (clone: 11B11; Ebiosciences, San Diego, CA), TNF-α (clone: MP6-XT22; Ebiosciences, San Diego, CA), and IFN-γ (clone: XMG1.2; BD Biosciences, San José, CA). Cells were incubated for an additional 60 minutes at RT, washed, and resuspended in BD Perm/Wash. The analysis of cytokines was done with an LSR-Fortessa (BD Biosciences San José, CA).

Poteet E., Lewis P., Li F., Zhang S., Gu J., Chen C., Ho S.O., Do T., Chiang S., Fujii G, & Yao Q. (2015). A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV. PLoS ONE, 10(8), e0136862.

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