Alexa488-conjugated anti-GATA3, Alexa647-conjugated anti-CXCR5, APC-Cy7-conjugated anti-CD4, BUV395-conjugated anti-IFNγ, BV711-conjugated anti-IL-2, Pe-Cy7-conjugated anti-CD25, PE-conjugated anti-CCR6 and anti-mouse IgG1, and PerCpCy5.5-conjugated anti-CD127 and anti-Tbet were purchased from Becton Dickinson. Alexa488-conjugated anti-IL-10, eFluor660-conjugated anti-IL-21, FITC-conjugated anti-CD45RA, PE-conjugated IL-22, Pe-Cy7-conjugated anti-IL-4 and mouse IgG1 were purchased from eBiosciences. APC-Cy7-conjugated anti-IL-17A, BV421-conjugated anti-CXCR3, and BV605-conjugated anti-TNFα was purchased from Biolegend. FITC-conjugated anti-CCR7 and recombinant human IL-12 was purchased from R&D Systems. Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology. Recombinant human TGFβ, IL-1β, IL-6, IL-21 and IL-23 were from Peprotech. Prostaglandin E2, PMA, calcium ionophore (ionomycin), Brefeldin A, and saponin were purchased from Sigma-Aldrich and recombinant human IL-4 was provided by Dr Rene de Waal Malefyt (DNAX Research Institute, Palo Alto, CA). T cell activation and expansion (TAE) beads (anti-CD2/CD3/CD28) were purchased from Miltenyi Biotec and CFSE was purchased from Invitrogen.
Calcium ionophore ionomycin
Manufactured by Merck Group
Calcium ionophore ionomycin is a laboratory reagent used to increase the concentration of calcium ions (Ca2+) within cells. It functions by facilitating the transport of calcium ions across cell membranes, leading to an elevated intracellular calcium level. This property makes ionomycin a useful tool in cell biology research for studying calcium-dependent cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
Apc cy7 conjugated anti cd4, by BD (1 mentions)
Pe cy7 conjugated anti cd25, by BD (1 mentions)
Pe conjugated anti ccr6, by BD (1 mentions)
Pe cy7 conjugated anti il 4, by Thermo Fisher Scientific (1 mentions)
Apc cy7 conjugated anti il 17a, by BioLegend (1 mentions)
Fitc conjugated anti ccr7, by R&D Systems (1 mentions)
Recombinant human il 12, by R&D Systems (1 mentions)
Tae beads anti cd2 cd3 cd28, by Miltenyi Biotec (1 mentions)
Il 23, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Il 21, by Thermo Fisher Scientific (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
Recombinant human tgf β, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Indo 1 am, by Thermo Fisher Scientific (1 mentions)
3 protocols using calcium ionophore ionomycin
1
Multiparameter Immunophenotyping of T Cells
2
Cytosolic Calcium Flux Quantification in MCF-7 Cells
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To measure cytosolic Ca2+, MCF-7 cells were stained with 1 μM Indo-1-AM (Thermo Fisher Scientific) in RPMI1640 medium (phenol-red free) containing 10% FBS at 37 °C for 45 min. Then, cells were washed and incubated for another 45 min at 37 °C in RPMI 1640 containing 10% FBS. After washing, the samples were analyzed using a flow cytometer (LSR I BD Bioscience, Heidelberg, Germany). Cells were illuminated with the 325 nm laser line of a helium–cadmium laser. Flourescence emissions at 390 to 420 nm and 500 to 520 nm were detected simultaneously, and changes in the ratio of the two emission intensities were analyzed with FLowJo software. To demonstrate successful loading with the dye, we induced maximal Ca2+ release by adding calcium ionophore ionomycin (10 mg/mL) (Sigma-Aldrich). Ca2+ efflux in MCF-7 cells was performed in response to varying concentrations of G1 or TG. DMSO was used as a negative control.
3
Isolation and Cytokine Induction of Liver Lymphocytes
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Liver infiltrating lymphocytes and PBMCs were thawed in PBS with 10 μg/mL DNase I (Sigma-Aldrich, St. Louis, MO) to prevent cell clumping. The cells were resuspended in RPMI 1640 medium (Gibco, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (Bioconcept, 4123 Allschwil, CH), 100 U/mL kanamycin, 2 mmol/L stable glutamine, 1 mmol/L sodium pyruvate, and 1% minimal essential medium (MEM) nonessential amino acids (all from Bioconcept), and 10 μg/mL DNase I. The cells were rested for 1 hour at 37°C before further processing. For intracellular cytokine induction, the cells were washed and resuspended in complete medium containing PMA (25 ng/mL), calcium ionophore ionomycin (1 μg/mL), and Brefeldin A (10 μg/mL; all from Sigma-Aldrich) for 4 hours. The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Norcross, GA) was used to identify viable cells before intracellular staining.
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