Ionomycin

Manufactured by Alexis Biochemicals

Sourced in United States

Ionomycin is a laboratory reagent used as a calcium ionophore. It functions by increasing intracellular calcium levels in cells, which can be used to study calcium-dependent cellular processes.

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5 protocols using ionomycin

Multiparametric Flow Cytometry Analysis of T-cell Subsets

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Peripheral blood mononuclear (PBMC) was isolated by Ficoll-Hypaque density centrifugation (Amersham Biosciences, UK). PBMCs were suspended at a density of 2 × 10⁶ cells/mL in Roswell Park Memorial Institute (RPMI) media 1640 with GlutaMAX (Gibco, USA).
For analysis of Th9/Th1/Th2/Th17/Treg cells, 2 × 10⁶ PBMC cells were seeded in 24-well plates and were stimulated with phorbol myristate acetate (PMA, 50 ng/mL) plus ionomycin (1 µM, all from Alexis Biochemicals, San Diago, CA, USA) and monensin (500 ng/mL, from eBioscience, San Diago, CA, USA) for 5 hours. The cells were collected and stained with antibodies against APC-CD3 and PE-Cy5- at 4°C for 30 minutes. After surface staining, the cells were fixed and permeabilized, followed by incubation with antibodies against IL-9-PE, IFN-γ-PE-cy7, IL-4-PE-cy7, Foxp3-PE-cy7, and IL-17-PE-cy7 for 30 minutes. Isotype-matched controls were used to correct nonspecific binding. All of the antibodies and reagents were purchased from eBioscience (San Diego, CA, USA). In the flow cytometry, the cells were gated on the forward scattering of living cells and then centered on CD3⁺CD4⁺T cells. All stained cells were analyzed by flow cytometry (ASR II) and FlowJo software (Tristar, USA). At least 50,000 events per samples were analyzed.

Pang N., Zhang F., Ma X., Zhang Z., Zhao H., Xin Y., Wang S., Zhu Y., Wen H, & Ding J. (2014). Th9/IL-9 Profile in Human Echinococcosis: Their Involvement in Immune Response during Infection by Echinococcus granulosus. Mediators of Inflammation, 2014, 781649.

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Multicolor Flow Cytometry for T Cell Subsets

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Cells were allocated into tubes and washed once in phosphate buffered saline (PBS). For Tregs analysis, the cells were incubated with anti-CD4-FITC-hAb and anti-CD25-APC-hAb (BD Pharmingen). After the surface staining, the cells were stained with anti-Foxp3-PE-hAb (BD Pharmingen) after fixation and permeabilization according to the manufacturer's instructions. For analysis of Th1, Th2, and Th17 cells, the cells were stimulated with phorbol myristate acetate (PMA, 20 ng/mL, Alexis Biochemicals, San Diego, CA) and ionomycin (1 μg/mL, Alexis Biochemicals) for 4 h in the presence of 2 μmol/mL monensin (Alexis Biochemicals). The incubator was set at 37°C under a 5% CO₂ environment. After culture for 4 hours, the cells were collected for staining according to the instructions. Fixation and permeabilization were necessary before staining with anti-IFN-γ-PE-, anti-IL-4-PE-, or anti-IL-17-PE- hAb (BD Pharmingen).

Zhu R., Liu C., Tang H., Zeng Q., Wang X., Zhu Z., Liu Y., Mao X, & Zhong Y. (2015). Serum Galectin-9 Levels Are Associated with Coronary Artery Disease in Chinese Individuals. Mediators of Inflammation, 2015, 457167.

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Expansion and Characterization of PBMCs and DCs

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Cell culture medium for PBMCs and DCs was RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS (Gibco, Carlsbad, CA, USA) and 100 U/mL streptomycin/penicillin. Anti-human CD3, anti-human CD28, anti-human CD4-FITC, anti-human CD25-APC, anti-human Foxp3-PE, anti-human IFN-γ-PE, anti-human IL-17-PE, anti-human CD11c-APC/Cy7, anti-human HLA-DR-PerCp/Cy5.5, anti-human CD40-PE, and anti-human CD86-PE were all from eBioscience, San Diego, CA, USA. TRIzol, PrimeScript RT reagent kit, and SYBR Green Master Mix were from Takara Biotechnology, Dalian, China. Recombinant human GM-CSF and recombinant human IL-4 were obtained from PeproTech, Rocky Hill, NJ, USA. Meanwhile, human magnetic CD14 isolation kit and CD4 isolation kit were bought from Miltenyi Biotec, Auburn, CA, USA. Lipopolysaccharides (LPS) were from Sigma-Aldrich, St Louis, MO, USA, and recombinant human IL-37 was bought from AdipoGen AG, Liestal, Switzerland. Oxidised low density lipoprotein (oxLDL) was acquired from Yiyuan, Wuhan, China, and phorbol myristate acetate, ionomycin, and monensin were from Alexis Biochemicals, San Diego, CA, USA.

Mao X., Zhu R., Zhang F., Zhong Y., Yu K., Wei Y., Sun H., Xu W., Luo Q., Wang Y., Ding Y, & Zeng Q. (2019). IL-37 Plays a Beneficial Role in Patients with Acute Coronary Syndrome. Mediators of Inflammation, 2019, 9515346.

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Evaluating Tc17 Cells in Blood

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To analyze the prevalence of Tc17 cells, IL-17-producing CD8(+) lymphocytes were evaluated by flow cytometry. In brief, heparinized peripheral whole blood (200 µl) with an equal volume of RPMI-1640 medium was incubated for 4 h at 37°C in 5% CO₂ in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 µg/ml of ionomycin, and 1.7 µg/ml monensin (all from Alexis Biochemicals, San Diego, CA). Then, the cells were incubated with PE-Cy5-conjugated anti-human CD3 and FITC-conjugated anti-human CD8 monoclonal antibodies (eBioscience, San Diego, CA) at room temperature in the dark for 15 min to stain the surface. After fixation and permeabilization, according to the manufacturer's instructions, the cells were stained with a PE-conjugated anti-IL-17 monoclonal antibody for 15 min. Isotype controls were used to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software (BD Bioscience PharMingen).

Zhang Y., Hou F., Liu X., Ma D., Zhang Y., Kong B, & Cui B. (2014). Tc17 Cells in Patients with Uterine Cervical Cancer. PLoS ONE, 9(2), e86812.

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Identification of Th22 Cells by Flow Cytometry

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Flow cytometry was used to study membrane makers and intracellular cytokines to identify the cytokine-producing cells. Briefly, heparinized peripheral blood with an equal volume of 1640 medium (Hyclone, USA) was incubated for 4 h at 37°C, 5% CO2 in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 μg/ml of ionomycin, and 1.7 μg/ml monensin (all from Alexis Biochemicals, USA). After incubation, the cells were stained with Alexa Fluor^® 647 or PerCP/Cy 5.5 anti-CD4 monoclonal antibody at room temperature in the dark for 20 min. After surface staining, the cells were next stained with FITC anti-IFN-γ, PerCP/Cy 5.5 or PE anti-IL-17 and PE or Alexa Fluor^® 660 anti-IL-22 monoclonal antibodies after fixation and permeabilization. All the antibodies were purchased from Biolegend and eBioscience (California, USA). Fixation and permeabilizaton reagents were purchased from eBioscience (California, USA). Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACS Calibur cytometer equipped with CellQuest software (BD Bioscience PharMingen, USA). For analysis, we first gated lymphocytes, then gated CD4⁺IFN-γ⁻ T cells in lymphocytes, and analyzed the percentages of CD4⁺IFN-γ⁻IL-17⁻IL-22⁺ (Th22) cells in CD4⁺IFN-γ⁻ T cells.

Lu T., Liu Y., Yu S., Yin C., Li P., Ye J., Ma D, & Ji C. (2016). Increased frequency of circulating Th22 cells in patients with B-cell non-Hodgkin's lymphoma. Oncotarget, 7(35), 56574-56583.

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