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Ionomycin

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Ionomycin is a calcium ionophore that facilitates the movement of calcium ions across cell membranes. It is commonly used in biomedical research as a tool to study calcium-mediated cellular processes.

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Lab products found in correlation

Brefeldin a, by BioLegend (51 mentions) Phorbol myristate acetate (pma), by BioLegend (19 mentions) Monensin, by BioLegend (11 mentions) Cell activation cocktail, by BioLegend (8 mentions) Phorbol myristate acetate (pma), by Merck Group (7 mentions) Ionomycin, by Merck Group (7 mentions) Golgiplug, by BD (5 mentions) Cytofix cytoperm kit, by BD (4 mentions) Ic fixation buffer, by Thermo Fisher Scientific (4 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions) Il 18, by Thermo Fisher Scientific (3 mentions) Il 12, by Thermo Fisher Scientific (3 mentions) Anti nk1, by BioLegend (3 mentions) Anti nkp46, by BioLegend (3 mentions) Hil 15, by Thermo Fisher Scientific (3 mentions) Facscalibur, by BD (3 mentions) Permeabilization buffer, by Thermo Fisher Scientific (3 mentions) Il 15, by R&D Systems (3 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions) Phorbol 12 myrstate 13 acetate pma, by BioLegend (3 mentions)

Close spelling variants of this product

PMA/ionomycin (32 citations) PMA/Ionomycin with Brefeldin A (4 citations) PMA/Ionomycin cocktail (3 citations) Ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (2 citations) Ionomycin Cell Activation Cocktail (2 citations) PMA/ionomycin/Brefeldin A (2 citations) PMA and ionomycin (2 citations) PMA/Ionomycin and Brefeldin A (2 citations) Cell Activation Cocktail PMA/Ionomycin (2 citations) PMA/ionomycin activation cocktail (2 citations)

80 protocols using ionomycin

1

Cytokine Stimulation and CyTOF Profiling

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Suspension mass cytometry (CyTOF) experiments were performed as previously described25 (link). Briefly, for cytokine stimulation, single-cell samples were stimulated using a pre-mixed cocktail of PMA, ionomycin, and brefeldin A (Biolegend) for 2.5 hours at 37°C. All samples were stained for viability using palladium (Sigma) for 5 minutes at RT and quenched with complete RPMI media. Each batch of up to 10 samples were live-cell barcoded using a 5-choose-3 scheme based on CD45 antibodies tagged with 5 different isotopically enriched metals at room temperature (RT) for 10 minutes. Multiplexed batches were Fc blocked (Invitrogen) and then stained with the antibodies purchased from indicated sources and used at described dilutions (Supplementary Table 4). Chemokine receptor stains were performed first at 37°C for 10 minutes followed by all other surface markers at RT for an additional 20 minutes. Intracellular markers were stained with Cytofix/Cytoperm Kit (BD) as per manufacturer’s protocol. Stained cells were fixed with 1.6% paraformaldehyde (ThermoFisher) in PBS and stored up to 1 week at 4°C. On the day before data acquisition, all cells were stained with rhodium Cell-ID (Fluidigm). All data was acquired using Helios™ at University of Maryland School of Medicine Center for Innovative Biomedical Resources Flow Cytometry and Mass Cytometry Core Facility, Baltimore, Maryland.
Ho W.J., Zhu Q., Durham J., Popovic A., Xavier S., Leatherman J., Mohan A., Mo G., Zhang S., Gross N., Charmsaz S., Lin D., Quong D., Wilt B., Kamel I.R., Weiss M., Philosophe B., Burkhart R., Burns W.R., Shubert C., Ejaz A., He J., Deshpande A., Danilova L., Stein-O’Brien G., Sugar E.A., Laheru D.A., Anders R.A., Fertig E.J., Jaffee E.M, & Yarchoan M. (2021). Neoadjuvant cabozantinib and nivolumab convert locally advanced hepatocellular carcinoma into resectable disease with enhanced antitumor immunity. Nature cancer, 2(9), 891-903.
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2

Phenotypic Analysis of T Cell Subsets

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For phenotypic analysis, single-cell suspensions were stained with specific antibodies against CD4 (GK1.5), CD25 (PC61), TIM-3 (5D12), and CTLA-4 (UC10-4F10-11) for 30 min at 4°C in the dark. Then cells were fixed and permeabilized using a Transcription Factor Buffer Set (BD Biosciences, San Diego, CA) and stained with specific antibodies against Foxp3 (FJK-16s) according to the manufacturer’s instructions. To determine cytokine interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1) expression, isolated cells were stimulated with PMA, ionomycin, and Brefeldin A (BioLegend, San Diego, CA) at 37°C for 6 hours. Then cells were washed and labeled with CD4 (GK1.5) and CD25 (PC61) for 30 min at 4°C in the dark. Intracellular staining for Foxp3 (FJK-16s), IL-10 (JES5-16E3), and TGF-β1 (TW7-16B4) was performed using a Transcription Factor Buffer Set. All antibodies were purchased from BD Biosciences, eBioscience, and BioLegend. After staining, cells were washed twice and resuspended in PBS. specimens were detected by the BD FACS Canto II system (BD Biosciences, USA) and data were analyzed with FlowJo 10.5 software (TreeStar, San Carlos, CA).
Gao Y.F., Fan X.Z., Li R.S, & Zhou X.S. (2022). Rapamycin relieves lupus nephritis by regulating TIM-3 and CD4+CD25+Foxp3+ Treg cells in an MRL/lpr mouse model. Central-European Journal of Immunology, 47(3), 206-217.
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3

Cytokine Production Profiling Assay

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Cells were stimulated phorbol 12-myristate-13-acetate (PMA) (81 nM) and ionomycin (1.3 μM) (Biolegend) for one hour at 37°C and 5% CO2 then monensin (2 μM) (Biolegend) was added without washing the cells for four additional hours. Cells were then washed with staining buffer, stained for surface receptors, then fixed for 20 minutes, and stained for intracellular cytokines in permeabilization buffer. The intracellular antibodies that were used were anti-TNF-α (clone MP6-XT22, Biolegend), anti-IL-6 (clone MP5-20F3, Biolegend), anti-IL-17A (clone TC11-18H10.1, Biolegend), anti-IFN-γ (clone XMG1.2, Biolegend), anti-IL-2 (clone JES6-5H4, Biolegend), anti-IL-4 (clone 11B11, Biolegend), and anti-IL-10 (clone JES5-16E3, Biolegend).
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII or Celesta (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).
Muhammad F., Wang D., McDonald T., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2020). TIGIT+ A2Ar-Dependent Anti-Uveitic Treg Cells are a Novel Subset of Tregs Associated with Resolution of Autoimmune Uveitis. Journal of autoimmunity, 111, 102441.
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4

Th9 Cell Differentiation Assay

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Cells (106/ml) were incubated in 96 well round-bottom plates in media alone (unstimulated) or stimulated in media containing PMA (50ng/ml, Sigma) and ionomycin (1μg/ml, Sigma) with monensin (2μM, Sigma) added at various time points during the 5.5–6 h stimulation at 37°C before analysis as previous described (12 (link)). In experiments where Th9 cells derived from WT and IL-2-deficient mice were co-cultured, PMA/ionomycin stimulation occurred in Eppindorf tubes under rotation at 37°C. Blocking antibodies to CD25 (3C7, 1μg/ml, Biolegend) were added 15 min prior to PMA/ionomycin stimulation and blocking antibodies to IL-2 (JES6-1A12 or S4B6-1, Biolegend or BioXcell), TNFα (MP6-XT22, Biolegend) and IL-4 were added (all antibodies used at 20 μg/ml) during stimulation as indicated. Real time PCR for analysis of gene expression, pSTAT protein staining, and retroviral transduction of primary cells was performed using standard methods as previously described (12 (link)).
Olson M.R., Ulrich B.J., Hummel S.A., Khan I., Meuris B., Cherukuri Y., Dent A.L., Janga S.C, & Kaplan M.H. (2017). Paracrine IL-2 is required for optimal type 2 effector cytokine production. Journal of immunology (Baltimore, Md. : 1950), 198(11), 4352-4359.
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5

Lymphocyte Surface Staining and Intracellular Cytokine Analysis

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MLNs were gently pushed through a 40-μM filter to obtain lymphocytes. Surface staining for lymphocytes was done in sterile 1× Hank’s balanced salt solution (HBSS) (Corning) supplemented with10 mMHepes (Cellgro), 2 mM EDTA (Cellgro), and 0.5% (v/v) fetal bovine serum (FBS) (Gibco BRL) for 20 min at 4°C. Cells were then washed twice in supplemented 1× HBSS and enumerated via flow cytometry. The following antibodies were used: anti-CD4 (FITC) and anti-CD3 (Pacific Blue).
For intracellular staining, cells were first stimulated with ionomycin (500 ng/ml), phorbol 12-myristate 13-acetate (5 ng/ml), and brefeldin A (5 mg/ml; BioLegend) for 4 hours at 37°C. Cells were surface-stained, washed, and then permeabilized and fixed in 100 ml of Perm/Fix buffer (eBioscience) overnight at 4°C. Cells were washed twice in Perm/Wash buffer (eBioscience) and then stained for intracellular cytokines with the following antibodies: anti–IFN-γ (phycoerythrin) and anti–IL-17A (allophycocyanin). These data were collected with a BD LSRFortessa and analyzed with FlowJo software.
Chiaro T.R., Soto R., Stephens W.Z., Kubinak J.L., Petersen C., Gogokhia L., Bell R., Delgado J.C., Cox J., Voth W., Brown J., Stillman D.J., O’Connell R.M., Tebo A.E, & Round J.L. (2017). A member of the gut mycobiota modulates host purine metabolism exacerbating colitis in mice. Science translational medicine, 9(380), eaaf9044.
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6

Cytokine Stimulation and CyTOF Profiling

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Suspension mass cytometry (CyTOF) experiments were performed as previously described25 (link). Briefly, for cytokine stimulation, single-cell samples were stimulated using a pre-mixed cocktail of PMA, ionomycin, and brefeldin A (Biolegend) for 2.5 hours at 37°C. All samples were stained for viability using palladium (Sigma) for 5 minutes at RT and quenched with complete RPMI media. Each batch of up to 10 samples were live-cell barcoded using a 5-choose-3 scheme based on CD45 antibodies tagged with 5 different isotopically enriched metals at room temperature (RT) for 10 minutes. Multiplexed batches were Fc blocked (Invitrogen) and then stained with the antibodies purchased from indicated sources and used at described dilutions (Supplementary Table 4). Chemokine receptor stains were performed first at 37°C for 10 minutes followed by all other surface markers at RT for an additional 20 minutes. Intracellular markers were stained with Cytofix/Cytoperm Kit (BD) as per manufacturer’s protocol. Stained cells were fixed with 1.6% paraformaldehyde (ThermoFisher) in PBS and stored up to 1 week at 4°C. On the day before data acquisition, all cells were stained with rhodium Cell-ID (Fluidigm). All data was acquired using Helios™ at University of Maryland School of Medicine Center for Innovative Biomedical Resources Flow Cytometry and Mass Cytometry Core Facility, Baltimore, Maryland.
Ho W.J., Zhu Q., Durham J., Popovic A., Xavier S., Leatherman J., Mohan A., Mo G., Zhang S., Gross N., Charmsaz S., Lin D., Quong D., Wilt B., Kamel I.R., Weiss M., Philosophe B., Burkhart R., Burns W.R., Shubert C., Ejaz A., He J., Deshpande A., Danilova L., Stein-O’Brien G., Sugar E.A., Laheru D.A., Anders R.A., Fertig E.J., Jaffee E.M, & Yarchoan M. (2021). Neoadjuvant cabozantinib and nivolumab convert locally advanced hepatocellular carcinoma into resectable disease with enhanced antitumor immunity. Nature cancer, 2(9), 891-903.
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7

IFN-γ Production Measurement Protocol

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For the measurement of IFN-γ production, splenocytes were activated in 96 well plates with hIL-15 (50 ng/ml), IL-12 (10 ng/ml, Peprotech), IL-18 (100 ng/ml), PMA (10 ng/ml), Ionomycin (1 μg/ml), or coated with anti-NK1.1 (25 μg/ml, BioLegend), anti-NKp46 (10 μg/ml, BioLegend), or LY49H (10 μg/ml, BioLegend). Cells were incubated with monensin and brefeldin A (BD GolgiPlug and GolgiStop) in complete medium for 4 h at 37°C. The cells were subjected to surface staining and intracellular staining was performed by use of the FoxP3/Transcription Factor Staining Buffer Set (eBioscience).
Delconte R.B., Guittard G., Goh W., Hediyeh-Zadeh S., Hennessy R.J., Rautela J., Davis M.J., Souza-Fonseca-Guimaraes F., Nunès J.A, & Huntington N.D. (2020). NK Cell Priming From Endogenous Homeostatic Signals Is Modulated by CIS. Frontiers in Immunology, 11, 75.
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8

Stimulation and Analysis of dLN Cells

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dLN cells harvested from immunized mice were aliquoted 1 × 106 cells/well in 96-well round-bottom cell culture plate (SPL Life Sciences, Pocheon-si, Korea). The cells were then stimulated with 1 μg/mL G4NA in complete RPMI (RPMI 1640 supplemented with 10% FBS and 1% of 1M HEPES) at 37 °C in a humidified 5% CO2 incubator for 16 h. A cell activation cocktail (phorbol 12-myristate-13-acetate and ionomycin, BioLegend) was used as a positive control. After the incubation, GolgiPlug (brefeldin A, BD Biosciences) and GolgiStop (Monensin, BD Biosciences) were added and incubated for four hours. Cells were then stained for flow cytometry analysis.
Kim D.B., Lee S.M., Geem K.R., Kim J., Kim E.H, & Lee D.W. (2022). In planta Production and Validation of Neuraminidase Derived from Genotype 4 Reassortant Eurasian Avian-like H1N1 Virus as a Vaccine Candidate. Plants, 11(21), 2984.
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9

Isolation and Characterization of Intestinal ILC3s

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Mice were sacrificed, and intestines were removed, opened longitudinally, and cut into 1cm pieces. The pieces were then incubated twice in 5 mM EDTA in PBS for 15 min at 37°C, and then the epithelial cell layer was removed by vortexing and passing through a 100-µm cell strainer. After incubation with EDTA solution, tissues were washed, minced into small pieces, and digested for 1 h at 37°C in digestion solution containing 4% FBS, 0.5 mg/ml collagenase D (Roche), 0.5 mg/ml DNase I (Sigma-Aldrich), and 3 mg/ml dispase II (Sigma-Aldrich). The resulting solution was strongly vortexed and passed through a 40-µm cell strainer. Mononuclear cells were isolated by gradient centrifugation on a 30/70% Percoll gradient (GE Healthcare) at 670 g for 30 min. Isolated cells were stained for ILC3 isolation or stimulated with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend) for 4 h and then stained with PerCP/Cy5.5-conjugated IL-17A, and APC-conjugated anti-CD4 antibodies. For ILC3 isolation, cells were stained with FITC-conjugated anti-Lineage cocktail, APC-conjugated anti-CD117, and PerCP/Cy5.5-conjugated anti-NKp46 antibodies, and then Lineage− NKp46− CD117+ cells were sorted. All fluorochrome-conjugated antibodies were purchased from BioLegend.
Kim H.S., Jang S.W., Lee W., Kim K., Sohn H., Hwang S.S, & Lee G.R. (2017). PTEN drives Th17 cell differentiation by preventing IL-2 production. The Journal of Experimental Medicine, 214(11), 3381-3398.
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10

Analyzing Intracellular Cytokines in Lamina Propria Lymphocytes

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For analysis of intracellular T cell cytokines (IL-10, IFNγ, IL-17A, IL-22), lamina propria lymphocytes were restimulated in complete RPMI with 5 ng/mL phorbal 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin in the presence of monensin (Biolegend) for 3.5 hours at 37°C. For analysis of intracellular ILC cytokines, lamina propria lymphocytes were incubated at 37°C for 4 hours with 5 mg/mL Brefeldin A (Biolegend) without additional restimulation. Dead cells were excluded from all analyses using Zombie Aqua Fixable Viability dye (Biolegend). For intracellular cytokine staining, cells were fixed with IC Fixation Buffer (eBioscience) and transcription factors were detected in unstimulated cells fixed with FoxP3 Fixation/Permeabilization buffers (eBioscience). Simultaneous detection of cytokines and transcription factors was achieved by sequential staining; first of cytokines using IC Fixation Buffer followed by transcription factors in Fixation/Permeabilization buffers. All data was acquired on the same LSRII instrument (BD Biosciences), with the exception of the data in Figure S3B that was acquired on a FACSAriaII (BD Biosciences), and analyzed using FlowJoX (TreeStar).
Britton G.J., Contijoch E.J., Mogno I., Vennaro O.H., Llewellyn S.R., Ng R., Li Z., Mortha A., Merad M., Das A., Gevers D., McGovern D.P., Singh N., Braun J., Jacobs J.P., Clemente J.C., Grinspan A., Sands B.E., Colombel J.F., Dubinsky M.C, & Faith J.J. (2019). Microbiotas from Humans with Inflammatory Bowel Disease Alter the Balance of Gut Th17 and RORγt+ Regulatory T Cells and Exacerbate Colitis in Mice. Immunity, 50(1), 212-224.e4.
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