Delphinidin chloride was purchased from Extrasynthèse (Genay, France) and was used at 10−2 g/L. This concentration has been described to induce the maximal relaxing effect on ex vivo rat aorta13 (link), to prevent angiogenesis through an inhibition of migration and proliferation16 (link), 17 (link) and to inhibit endothelial apoptosis51 (link). Delphinidin was diluted in dimethylsulfoxide (DMSO) from Sigma Aldrich (St Louis, MO). The final concentration of DMSO in experiments never exceeded 0.1%. Anti-CD3 (clone OKT3) and anti-CD28 (clone CD28.2) human antibodies were purchased from BioLegend® (San Diego, CA). Histopaque®1077, Histopaque®1083, thapsigargin, Phytohemagglutinin (PHA), phorbol-12-myristate-13-acetate (PMA), ionomycin, fulvestrant, and SKF96365 were purchased from Sigma-Aldrich. Mibefradil hydrochloride was purchased from Abcam (Cambridge, UK) and trichostatin A (TSA) from Santa Cruz Biotechnology (Santa Cruz, CA). U0126 was obtained from Calbiochem (San Diego, CA). RPMI-1640, Na-pyruvate, non-essential amino-acid (NEAA) and penicillin/streptomycin were purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) and Fluo-4 acetoxymethyl ester (AM) were purchased from Life Technologies (Carlsbad, CA).
Na pyruvate
Manufactured by Lonza
Sourced in Belgium
Na-pyruvate is a laboratory reagent used as a cell culture supplement. It serves as a source of pyruvate, an important metabolic intermediate in cellular respiration and energy production processes. The core function of Na-pyruvate is to provide a readily available source of pyruvate for cells in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Lonza (2 mentions)
Rpmi 1640, by Lonza (2 mentions)
Non essential amino acid solution, by Lonza (2 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Lonza (2 mentions)
Histopaque 1083, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Anti cd3 clone okt3, by BioLegend (1 mentions)
Trichostatin a tsa, by Santa Cruz Biotechnology (1 mentions)
Phorbol 12 myristate 13 acetate pma ionomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Non essential amino acid (neaa), by Lonza (1 mentions)
Anti cd28 clone cd28, by BioLegend (1 mentions)
Fulvestrant, by Merck Group (1 mentions)
Delphinidin, by Merck Group (1 mentions)
Skf96365, by Merck Group (1 mentions)
Delphinidin chloride, by Extrasynthese (1 mentions)
8 protocols using na pyruvate
1
Delphinidin Chloride: Molecular Insights and Therapeutic Potential
2
Prostate Cancer Cell Line Cultivation
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Human cell lines PC3, DU145, and LNCaP were obtained from American Type Culture Collection (ATCC, Rockville, MD). LAPC4 were a kind gift from Prof. A. Cato (Institute of Toxicology and Genetics, Karlsruher Institut für Technologie, Germany). Docetaxel‐resistant PC3‐DR and DU145‐DR were previously established by Puhr et al.14 All cells were cultured in Roswell Park Memorial Institute 1640 (RPMI‐1640) (PAN Biotech, Aidenbach, Germany) containing 10% (v/v) fetal calf serum (PAN Biotech), 1% (v/v) penicillin/streptomycin and 1% (v/v) GlutaMAX (both from Lonza, Vienna, Austria). LNCaP were supplemented with 1% (v/v) 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) (Sigma, Vienna), 1% (v/v) d ‐glucose (Sigma), 1% (v/v) Na‐pyruvate (Lonza) and LAPC4 with 100 nmol/L dihydrotestosterone (Sigma). PC3‐DR and DU145‐DR were cultured in the presence of 12.5 nmol/L Docetaxel (Sigma). The authenticity of all cell lines was validated via short tandem repeat profiling.
3
ASFV Propagation in Porcine Alveolar Macrophages
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Porcine alveolar macrophage (PAM) cells were freshly harvested from swine lungs according to the OIE Manual’s instructions [27 ]. PAM cells were used for the propagation of the highly virulent ASFV_HU_2018 isolate of the African swine fever virus (Accession Number: MN715134.1). PAM cells were grown in RPMI 1640-containing L-glutamine (Lonza, Basel, Switzerland) medium supplemented with 10% fetal bovine serum (Euro Clone, Pero, Italy), 1% Na-pyruvate (Lonza), 1% non-essential amino acid solution (Lonza), and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO2 in air gas phase. The infectious titer of serially diluted viral stock was calculated using an immunofluorescence (IF) assay as described earlier [22 (link)]. PAMs were cultivated in 6-well plates at a density of 3.3 × 105 cells and infected at a multiplicity of infection (MOI) of 10 plaque-forming units per cell at 4 h after cell seeding. The supernatant was replaced with a fresh medium after 1 h post-infection (hpi). Infected PAM cells were harvested at 4, 8, 12, and 20 hpi, whereas mock-infected cells were harvested at 20 hpi IF assay was used for monitoring the efficiency of infection in an infected control well fixed at 20 hpi.
4
Endothelial Cell Culture and Pharmacological Modulation
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Human umbilical vein ECs (HUVECs) and ECs from healthy donor and patients were cultured in EGM‐2 (EBM‐2 + SingleQuots™ Kit) and 2% foetal bovine serum (FBS) (Lonza). HUVECs and ECs were used between P2 and P6 passages. HEK293T (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose, 2 mM l ‐glutamine, without Na pyruvate (Lonza), 10% FBS and 1% penicillin/streptomycin (Pen/Strep). Reagents and doses were as follows: LysoTracker (Thermo Fisher), bafilomycin A1 (Sigma, 200 nM), torin‐1 (CST, 10 μM), BAY11‐7082 (Abcam, 300 nM), TYRPHOSTIN AG1288 (Abcam, 300 nM), SB202190 (Tocris, 300 nM), cycloheximide (Sigma, 2 μM) and MG132 (Sigma, 2 μM).
5
Murine Bone Marrow-Derived Macrophage Differentiation
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C57BL/6 mice were purchased from Charles River (Charles River ITALY). BMDMs were derived from the bone marrow cells collected from five-week old female mice, as already reported [39 (link)]. Subsequently, BMDMs were differentiated during 5 days in RPMI1640 (Lonza, Basel, Switzerland), supplemented with 10% FBS (Hyclone, San Angelo, TX, USA), 1% Glutamine (Lonza, Basel, Switzerland), 1% Na pyruvate (Lonza, Basel, Switzerland), 1% NEAA (Lonza, Basel, Switzerland), 0.5% 2-ME (Thermo Fisher Scientific, Milan,, Italy), and 30 ng/mL macrophage colony-stimulating factor (M-CSF; Miltenyi Biotec, Bergisch Gladbach, Germany).
6
Culturing DU145 Prostate Cancer Cells
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The DU145 cell line was obtained from American Tissue Culture Collection (ATCC) (Manassas, VA, USA). DU145 cells were grown in Minimal Essential Medium (MEM) with the addition of 1% (v/v) non-essential amino acids (NEAA), 10 % (v/v) fetal bovine serum (FBS), 1% (v/v) Na-Pyruvate, 100 mg/mL streptomycin and 100 U/mL penicillin, bought from Lonza (Lonza Sales AG, Verviers, Belgium). Cells were incubated inside 175 cm2 flasks at 37 °C in a humidified 5% CO2 incubator, and after reaching confluence, detaching was obtained with the addition of 2 mL of Trypsin–EDTA solution.
7
Culturing Human Multiple Myeloma Cell Lines
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HMCLs were obtained from ATCC (Molsheim, France). RPMI-8226, OPM-2 and JJN3 were cultured in RPMI-1640 medium (Lonza, Basel, Switzerland) with 10% FCS (Biochrom AG, Berlin, Germany) and supplements (100U/ml penicillin/streptomycin and 2mM L-glutamine (Lonza)). NCI-H929 cells were grown in RPMI-1640 medium with 20% FCS (Biochrom AG), supplements plus 1mM Na-Pyruvate (Lonza) and 55µM β-mercaptoethanol (Sigma, Bornem, Belgium). Authenticity of HMCLs was regularly confirmed by short-tandem repeat analysis.
8
Protocol for ASFV Propagation in PAM Cells
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Pulmonary macrophage (PAM) cells were harvested freshly from swine lungs according to the OIE Manual's instructions [45] . PAM cells were used for the propagation of the highly virulent ASFV_HU_2018 isolate of African swine fever virus (ID Number: MN715134). PAM cells were grown in RPMI 1640 containing L-glutamine (Lonza) medium supplemented with 10% foetal bovine serum (Euro Clone), 1% Na-pyruvate (Lonza), 1% non-essential amino acid solution (Lonza), and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific) at 37°C in 5% CO 2 in air gas phase. The infectious titre of serially diluted viral stock was calculated using an immunofluorescence (IF) assay as described earlier [31] (link). PAMs were cultivated in 6-well plates at a density of 3.3×10 5 cells and infected at a multiplicity of infection (MOI) of 10 plaque-forming unit per cell at 4 h after cell seeding. Supernatant was replaced with fresh medium after 1 h post-infection (p.i.). Infected PAM cells were harvested at 4, 8, 12, and 20 h p.i., whereas cock-infected cells were harvested at 20 h p.i. IF assay was used for monitoring the efficiency of infection in an infected control well fixed at 20 h p.i. Infection efficiency remained at approximately 20% despite the high viral titre (MOI = 10) applied for the infection. This result is in agreement with other's observations.
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