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8 protocols using ionomycin calcium salt from streptomyces conglobatus

1

Neutrophil Ca2+ Signaling Assay

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Neutrophils (1 × 106/ml) were loaded with the Ca2+-sensitive fluorogenic dye FLUO-3/AM (2 µM; Molecular Probes, Eugene, Oreg., USA) at 37 °C for 30 min and washed twice. Stimuli were added as indicated, fluorescence signals were acquired by flow cytometry and analyzed with FlowJo software V8.7.1 (Tree Star Inc., Ashland, Oreg., USA)15 (link). Ionomycin calcium salt from Streptomyces conglobatus (1 µM; Sigma-Aldrich, Taufkirchen, Germany) served as positive control.
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2

Calcium Imaging of B16 Cells

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B16 cells cultured on 35-mm dishes were incubated in culture medium containing 3.5 μmol/L FURA-2-AM (Invitrogen) for 1 h at 37 °C, and then rinsed with Hanks’ Balanced Salt Solution (HBSS, Sigma). Each dish was placed into a culture chamber at 37 °C on the stage of an inverted fluorescence microscope (TE2000E, Nikon Instruments, Fi, Italy), connected to a cooled CCD camera (512B Cascade, Roper Scientific, Ottobrunn, Germany). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International, New Jersey, USA) and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image per s) using Metafluor software (Universal Imaging Corporation, Downington PA, USA). Calibration was obtained at the end of each experiment by maximally increasing intracellular Ca2+-dependent FURA-2AM fluorescence with 5 μmol/L ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal fluorescence in a Ca2+-free medium. [Ca2+]i was calculated according to the formulas previously described54 (link).
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3

Xenopus Egg Activation and Fertilization Imaging

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Xenopus eggs were obtained via established methods. Eggs were acquired from female Xenopus laevis (Nasco) between the ages of 3 to 10 years old. Confocal images were taken at 2.5X using a Leica SP5 microscope (Biological Imaging Facility, Northwestern University). Eggs were imaged in 0.1X Ca2+, Mg2+, and EDTA-free Marc’s Modified Ringer’s (MMR: 10 mM NaCl, 200 μM KCl, 500 μM HEPES pH 7.4) buffer containing 50 μM FluoZin-3 tetrapotassium salt ((ThermoFisher Scientific).and implement XFM experiments and process and analyze ) In order to parthenogenically activate the eggs, ionomycin calcium salt from Streptomyces conglobatus (Sigma Aldrich) in DMSO was added to the buffer at a final concentration of 20 μM. To image fertilization, around half of a testis was ground in 200 μl 1X Ca2+, Mg2+, and EDTA-free MMR (100 mM NaCl, 2 mM KCl, 5 mM HEPES pH 7.4) and kept on ice. Before imaging, the sperm solution was mixed 1:1 with 0.1X Ca2+, Mg2+, and EDTA-free MMR and added at a 1:10 dilution to the buffer in the imaging dish.
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4

Cellular Signaling Compounds Protocol

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Ionomycin calcium salt from Streptomyces conglobatus was purchased from SigmaAldrich (St Louis, MO), and Arachidonyl-2′-chloroethylamide (ACEA) and rimonabant from Tocris Bioscience (Bristol, UK).
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5

Comprehensive Immune Cell Phenotyping

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Surface markers on cells were stained with Abs in PBS with 2% bovine growth serum (FACS buffer) at 4°C for 20 min. For TFH staining, cells were stained with purified CXCR5 for 1 h in FACS buffer + 0.5% BSA + 2% normal mouse serum (NMS), followed by biotin-goat anti-rat IgG for 30 min in FACS buffer + 2% NMS, and then streptavidin and other surface-staining Abs for 30 min in FACS buffer + 2% NMS at 4°C. For intracellular cytokine/molecule detection, splenocytes were restimulated ex vivo with 2.5μg/ml gp15067–83 peptide (LSNNNPTTIMRPPVAQN) or 50ng/ml PMA (Sigma-Aldrich, Milwaukee, WI) and 1μg/ml ionomycin calcium salt from Streptomyces conglobatus (Sigma-Aldrich) in complete medium with 10U/ml rIL-2 and 10μg/ml brefeldin A (Sigma-Aldrich) at 37°C for 5h. Subsequently, cells were stained with Abs against surface markers for 20 min at 4°C, followed by fixation with 1% formaldehyde at 4°C for 20 min, and then stained with Abs against IFN-γ, TNF-α, IL-2 or GzmB in 0.5% saponin solution at 4°C for 30 min. For transcription factor detection, Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used.
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6

Measuring Intracellular Calcium Dynamics

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Cells were incubated in EGM-2 containing 3.5 µM Fura-2-AM (Invitrogen) for 1 h at 37 °C and then rinsed with either Hanks’ Balanced Salt Solution (HBSS, Sigma) or Ca2+ free Krebs Henseleit Hepes buffer (KHH). Dishes were placed into a culture chamber kept at 37 °C controlled temperature on the stage of an inverted microfluorimeter (Nikon TE2000E) connected to a cooled CCD camera (512B Cascade, Princeton Instruments). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International) and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image/s) using Metafluor software (Universal Imaging Corporation). Calibration of the signal was obtained at the end of each experiment by maximally increasing intracellular Ca2+-dependent Fura-2 fluorescence ratio (340/380) with 5 µM ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal ratio in Ca2+-free medium. [Ca2+]i was calculated according to previously described formulas55 (link).
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7

Xenopus Egg Activation and Fertilization Imaging

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Xenopus eggs were obtained via established methods. Eggs were acquired from female Xenopus laevis (Nasco) between the ages of 3 to 10 years old. Confocal images were taken at 2.5X using a Leica SP5 microscope (Biological Imaging Facility, Northwestern University). Eggs were imaged in 0.1X Ca2+, Mg2+, and EDTA-free Marc’s Modified Ringer’s (MMR: 10 mM NaCl, 200 μM KCl, 500 μM HEPES pH 7.4) buffer containing 50 μM FluoZin-3 tetrapotassium salt ((ThermoFisher Scientific).and implement XFM experiments and process and analyze ) In order to parthenogenically activate the eggs, ionomycin calcium salt from Streptomyces conglobatus (Sigma Aldrich) in DMSO was added to the buffer at a final concentration of 20 μM. To image fertilization, around half of a testis was ground in 200 μl 1X Ca2+, Mg2+, and EDTA-free MMR (100 mM NaCl, 2 mM KCl, 5 mM HEPES pH 7.4) and kept on ice. Before imaging, the sperm solution was mixed 1:1 with 0.1X Ca2+, Mg2+, and EDTA-free MMR and added at a 1:10 dilution to the buffer in the imaging dish.
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8

Splenocyte Activation Assay

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Spleens were mashed through a strainer to isolate spleen cells (splenocytes). Cells were then incubated for 3 hr with a mixture of 1 μg/ml Ionomycin calcium salt from Streptomyces conglobatus (56092-82-1, Sigma–Aldrich) and 1 ng/m phorbol 12-myristate 13-acetate (16561-29-8, Sigma-aldrich) to activate cells from naive T to mature T cells.
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