Nitric oxide (NO) release into the cell culture media was measured with a Nitric Oxide Analyzer (Sievers 270B) according to lab standards94 (link). The system measures nitrite, the major product of NO, which is generated when NO is exposed to superoxide anion. Therefore, NO that is produced in the media is able to be measured as nitrite after exposure to iodide and acetic acid at room temperature. Ionomycin from streptomyces conglobatus (Sigma) was added 1 h before collection to serve as a positive control. The media was collected for NO measures, and the cells were collected in protein lysis buffer to measure the protein level. All NO measurements were corrected for total protein (mg) based on whole cell lysates.
Ionomycin from streptomyces conglobatus
Manufactured by Merck Group
Sourced in Senegal
Ionomycin is a calcium ionophore derived from the bacterium Streptomyces conglobatus. It functions by increasing intracellular calcium levels in cells.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Annexin 5 conjugate, by Thermo Fisher Scientific (1 mentions)
Facscalibur system, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Lps from escherichia coli, by Merck Group (1 mentions)
Recombinant murine gm csf, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Ici 182 780, by Bio-Techne (1 mentions)
Cell culture medium, by Merck Group (1 mentions)
Phytohaemagglutinin pha, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
17β estradiol e2, by Merck Group (1 mentions)
Brefeldin a, by BD (1 mentions)
5 protocols using ionomycin from streptomyces conglobatus
1
Quantitative Nitric Oxide Measurement
2
Annexin V Staining for Erythrocyte Apoptosis
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Detection of erythrocyte apoptosis with Annexin V conjugates by flow cytometry (Annexin V conjugate, ThermoFisher scientific) was performed in a BD FACSCalibur™ system (BD Biosciences, San Jose, CA). Erythrocytes previously treated with ionomycin from Streptomyces conglobatus (Sigma-Aldrich) were used as a positive control for erythrocyte apoptosis. Erythrocytes derived from healthy animal blood with and without annexin were used as negative controls.
3
Preparation of Tumor-Targeting Vaccine VSSP
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VSSP was prepared at CIM by hydrophobic conjugation of the GM3 ganglioside with outer membrane vesicles from Neisseria meningitidis strain 385 (Finlay Institute, Havana, Cuba), as described elsewhere [26 (link)]. Chicken OVA grade VII, Con A from Canavalia ensiformes, LPS from Escherichia coli, PMA, Ionomycin from Streptomyces conglobatus and polyI:C were purchased from Sigma-Aldrich (Saint Louis, MO). OVA257-264 peptide (SIINFEKL) was synthetized at the Center for Genetic Engineering and Biotechnology (CIGB, Havana, Cuba) and CFSE was obtained from Invitrogen (Paisley, UK). Recombinant murine GM-CSF was purchased from PeproTech (NJ, USA). EL4 and EG.7 (EL4 cell line transfected with the gene encoding for OVA) lymphomas, and MCA203 sarcoma are derived from C57BL/6 mice (H-2b) and were kindly provided by Dr. Vincenzo Bronte. The metastatic clone of Lewis lung carcinoma cell line (3LL-D122) also syngeneic for C57BL/6 strain was obtained from the American Type Culture Collection (ATCC, Manassas, VA).
4
Immune Cell Stimulation Assay
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G (CAS #1071-83-6, purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). It was dissolved in Dulbecco’s Phosphate Buffered Saline. Phorbol 12-myristate 13-acetate (PMA – CAS #16561-29-8, purity ≥ 99%) and ionomycin from Streptomyces conglobatus (CAS #56092-81-0, purity ≥ 98%) were purchased from Sigma-Aldrich and used to stimulate immune cells, as well as phytohaemagglutinin (PHA, CAS #9008-97-3). 17β-estradiol (E2 – CAS #50-28-2, purity 98%) was used as reference compound for ER activity, and it was obtained from Sigma-Aldrich. ICI 182,780 (CAS # 129453-61-8, purity ≥ 99%) was used as ER antagonist and it was obtained from Tocris Bioscience (Bristol, United Kingdom). All the substances, with the exception of G, were dissolved in dimethyl sulfoxide (DMSO, CAS # 67-68-5, purity ≥ 99.5%), with a final DMSO concentration in culture medium ≤ 0.2%. Cell culture medium and all supplements were also purchased from Sigma-Aldrich.
5
Activation of Cytolytic γδ T Cells
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γδ T cells were stimulated with 100 ng/mL PMA (Sigma-Aldrich) and 1 µg/mL ionomycin (from Streptomyces conglobatus, Sigma-Aldrich) for 1.5 hours at 37°C and 5% CO2. Brefeldin A (1:1000, BD) was added to the medium at the start of stimulation for intracellular measurement of IFN-γ, TNF-α and granzyme B. For analysis, the cells were transferred to a V-bottom plate and washed with cold PBS/0.5%BSA prior to staining. Production of IFN-γ, TNF-α and granzyme B was measured using flow cytometry.
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