BMDMs plated as described above were incubated with Zymosan A from Saccharomyces cerevisiae (Sigma-Aldrich) at a ratio of 5,10 and 20 particles per BMDM for 1 h at 34°C, washed in PBS to remove non-internalized particles and reincubated for 48 h prior to the motility analysis.
Zymosan a from saccharomyces cerevisiae
Manufactured by Merck Group
Sourced in United States
Zymosan A is a cell wall preparation derived from the yeast Saccharomyces cerevisiae. It consists primarily of carbohydrates, including β-glucans and mannans. Zymosan A is commonly used as a research tool to study immune responses and inflammation in various in vitro and in vivo models.
Automatically generated - may contain errors
Lab products found in correlation
Luminol, by Merck Group (2 mentions)
Oxiselect intracellular ros assay kit, by Cell Biolabs (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (1 mentions)
Yeast extract peptone dextrose, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
D mannose, by Merck Group (1 mentions)
D mannose agarose, by Merck Group (1 mentions)
Dylight 488, by Thermo Fisher Scientific (1 mentions)
3 4 dihydroxy dl phenylalanine, by Merck Group (1 mentions)
L fucose, by AppliChem (1 mentions)
Methyl α l fucopyranoside, by Biosynth (1 mentions)
D glucose, by Biosynth (1 mentions)
D galactose, by Biosynth (1 mentions)
N acetyl d glucosamine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sb216763, by Selleck Chemicals (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Lps from escherichia coli, by InvivoGen (1 mentions)
Ic87114, by Selleck Chemicals (1 mentions)
27 protocols using zymosan a from saccharomyces cerevisiae
1
Phagocytosis Assay with Zymosan
2
Flow Cytometry Analyses: Microbial Stimuli
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Details about reagents, kits and antibodies used for flow cytometry analyses are reported in Supplementary Information
3
Zymosan-Activated Serum for PMN Assay
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A stock solution of zymosan-activated serum (ZAS) was prepared beforehand by incubating 10 mL of pooled blood serum from healthy cows with 100 mg of Zymosan A from Saccharomyces cerevisiae (Sigma-Aldrich, Winston Park, Oakville, ON, Canada) at 37 °C under gentle agitation for 60 min. After incubation, the activated serum was centrifuged at 390× g for 10 min, aliquoted and stored at −20 °C.
For every sample (blood and endometrium), two 1.5 mL microcentrifuge tubes were filled with 1 × 106 PMN suspended in 200 µL of RPMI-BSA, and 50 µL of ZAS was added to both tubes (PC+ and PC−). In the first tube (PC+), 1 µL fluorescently labeled 1 µm beads (FluoSpheres carboxylate, yellow-green (505/515), Life Technologies Corporation, Eugene, OR, USA) was added per 1 × 106 PMN, while no FluoSpheres were supplemented in the second tube (PC−, autofluorescence). Next, cells were incubated in the dark at 38.5 °C under gentle agitation for 30 min. After incubation, cells were washed and diluted in RPMI-BSA buffer (200 µL, 376 × g, 5 min, 10 °C), put on ice and protected from light until analysis.
For every sample (blood and endometrium), two 1.5 mL microcentrifuge tubes were filled with 1 × 106 PMN suspended in 200 µL of RPMI-BSA, and 50 µL of ZAS was added to both tubes (PC+ and PC−). In the first tube (PC+), 1 µL fluorescently labeled 1 µm beads (FluoSpheres carboxylate, yellow-green (505/515), Life Technologies Corporation, Eugene, OR, USA) was added per 1 × 106 PMN, while no FluoSpheres were supplemented in the second tube (PC−, autofluorescence). Next, cells were incubated in the dark at 38.5 °C under gentle agitation for 30 min. After incubation, cells were washed and diluted in RPMI-BSA buffer (200 µL, 376 × g, 5 min, 10 °C), put on ice and protected from light until analysis.
4
Saccharide Synthesis and Characterization
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Methyl-α-l -fucopyranoside, methyl-β-l -fucopyranoside, d- glucose and d- galactose were purchased from Carbosynth, Compton, United Kingdom; blood group A/B/O trisaccharides, and Fucα1-GlcNAc and Fucα1-GlcNAc were purchased from Carbohydrate Synthesis, Oxford, United Kingdom; l- fucose was purchased from Applichem, Darmstadt, Germany. N-acetyl-d -glucosamine, d -mannose, d -mannose-agarose, biotin and streptavidin, bovine serum albumin, 3,4-dihydroxy-dl -phenylalanine, zymosan A from Saccharomyces cerevisiae and luminol were purchased from Sigma-Aldrich, St Louis, USA. Biotinylated saccharides (biotinylated α/β-d -mannoside, α/β-d- galactoside and α-l -fucoside) were purchased from Synthaur LLC, Moscow, Russia. Fluorescein isothiocyanate (FITC) and DyLight 488 were purchased from ThermoScientific, Rockford, USA. Basic chemicals were purchased from Sigma-Aldrich, St Louis, USA; Duchefa, Haarlem, Netherlands; ForMedium, Hunstanton, United Kingdom and Applichem, Darmstadt, Germany.
5
Immune Stimulants and Inhibitors Protocol
6
Macrophage Respiratory Burst Assay
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Monocytes (N=5 cord blood samples) (105 cells/well) were incubated in black, clear bottom 96 well plates and differentiated into macrophages as described above. Macrophage respiratory burst was measured by using the OxiSelect™ Intracellular ROS Assay Kit (Cell Biolabs Inc., San Diego, CA). Differentiated macrophages were washed with PBS and pre-incubated for 1 h at 37°C with 10−8M IL-10, DEX or BETA, with PBS as a control. At 30 min, cell permeable fluorogenic probe 2′,7″-dichlorohydrofluoroescin diacetate (DCFH-DA) was added to the cell media. After 1 h cells were washed and stimulated with Zymosan A from Saccharomyces cerevisiae (0.5 mg/ml)(Sigma-Aldrich, St. Louis, MO). Cells incubated with hydrogen peroxide served as a positive control during the assay. The DCFH oxidized to highly fluorescent 2′7′-Dichlorohydrofluoroscein (DCF) by ROS generated during the incubation with zymosan, was read at 0 h (baseline) and 1 h, using a fluorescent plate reader with 480nm excitation and 530nm emission.
7
Chemicals and Reagents for Experimentation
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HNE was purchased from Calbiochem (San Diego, CA, USA). Genipin was purchased from Wako Pure Chemicals (Japan). Triphenyltetrazolium chloride (TTC) and Zymosan A from Saccharomyces cerevisiae were both purchased from Sigma-Aldrich (St. Louis, MO, USA). Phthalo blue dye was purchased from Quantum Ink Company (Louisville, KY, USA).
8
Quantifying Mouse Immune Mediators
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ELISA kits for mouse TNFα, IL-10, and IL-6 were obtained from Biolegend; a mouse CCL3 detection kit was obtained from R&D Systems. LPS (from Escherichia coli, clone 055:B5) and zymosan A (from Saccharomyces cerevisiae) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Docosahexaenoic acid (DHA) was purchased from Cayman Chemicals.
9
Chemotaxis Assays for Immune Cells
10
Zymosan-Induced Peritoneal Inflammation
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