Neutrophils (5 × 105 cells/100 μl) were preincubated for 30 min in bicarbonate- or HEPES-buffered medium (pH 7.4, 6.5, 6.0, and 5.5). Subsequently, Alexa-Fluor 488 conjugated opsonized non-viable Staphylococcus aureus bioparticles (Invitrogen; S. aureus to neutrophil ratio 2:1) or FluoSphere carboxylate-modified latex microspheres with a diameter of 1 µM [Invitrogen; final concentration of 0.015% (v/v)] were added and the co-culture was incubated for further 30 min. Cultures were placed on ice to stop phagocytosis, cells were washed to remove extracellular bacteria/beads, and trypan blue was added to quench fluorescence of extracellular bacteria/beads sticking on the neutrophil surface. Phagocytosis was assessed by flow cytometry using a FACS CantoII flow cytometer.
Staphylococcus aureus bioparticles
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Staphylococcus aureus BioParticles are a laboratory tool used for the detection and study of Staphylococcus aureus, a common bacterial pathogen. These particles are made up of formalin-fixed, whole Staphylococcus aureus cells and are used as a standardized reagent in various microbiological applications.
Automatically generated - may contain errors
5 protocols using staphylococcus aureus bioparticles
1
Neutrophil Phagocytosis Assay at Varying pH
2
Phagocytosis of S. aureus BioParticles
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Cells were incubated with or without fluorescein-conjugated Staphylococcus aureus BioParticles (Invitrogen, #S2851) at a ratio of 100 particles per cell for two hours at 37°C, washed twice with PBS/ 2 % BSA, resuspended in 300 μL FACS buffer and analyzed by flow cytometry.
3
Evaluation of Immune Cell Function
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Viability was assessed using CellTiter 96 AQueous One Solution (Roche, UK). Apoptosis was assessed by flow cytometry using an Annexin V assay (BD Biosciences, UK) in combination with the vital dye propidium iodide (PI) (Sigma-Aldrich, UK). CXCL-8, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α and matrix metalloproteinase (MMP)-9 levels in cell-free supernatants were quantified using commercially available ELISA kits (Biotechne, UK). Reactive oxygen species (ROS) were measured using DCFDA assay (Abcam ab113851) according to the manufacturer’s instructions. Phagocytosis assay was carried out using pHrodo Red Escherichia coli or Staphylococcus aureus BioParticles (Invitrogen, UK) according to the manufacturer’s instructions.
4
Opsonization and Phagocytosis Assay
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Staphylococcus aureus BioParticles conjugated to AlexaFluor 488 (Molecular Probes, Eugene, OR) were resuspended in PBS at 20 mg/ml and incubated in 20% participant plasma at 37°C for 1 hour. The bacteria were diluted 1:10 (v/v) in HBSS with calcium/magnesium. Neutrophils from a healthy donor (Astarte Biologics, Bothell, WA) were thawed in HBSS with calcium/magnesium and warmed at 37°C for 10 minutes. 5x105 neutrophils were incubated with 5x105 opsonized bacteria at 37°C for 10 minutes while rotating gently. Cells were placed on ice, fixed with cold 4% paraformaldehyde, and resuspended in HBSS (no calcium/magnesium). Fluorescence was measured on a Fortessa (BD Biosciences, San Jose, CA).
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Staphylococcus aureus BioParticles conjugated to AlexaFluor 488 (Molecular Probes, Eugene, OR) were resuspended in PBS at 20 mg/ml and incubated in 20% participant plasma at 37°C for 1 hour. The bacteria were diluted 1:10 (v/v) in HBSS with calcium/magnesium. Neutrophils from a healthy donor (Astarte Biologics, Bothell, WA) were thawed in HBSS with calcium/magnesium and warmed at 37°C for 10 minutes. 5x105 neutrophils were incubated with 5x105 opsonized bacteria at 37°C for 10 minutes while rotating gently. Cells were placed on ice, fixed with cold 4% paraformaldehyde, and resuspended in HBSS (no calcium/magnesium). Fluorescence was measured on a Fortessa (BD Biosciences, San Jose, CA).
5
Phagocytosis Assay for GMPs
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WT or TAZ-KO GMPs were differentiated out of E2 for a period of 4 days. Cells were resuspended in media with fluorescein-labeled heat-killed Escherichia coli or Staphylococcus aureus bioparticles (Molecular Probes) at a ratio of 1:3. Cells and bioparticles were agitated gently for 45 min at 37°C. Cells were washed in PBS and resuspended in FACS buffer for flow cytometry. Cells were also stained with mouse antibody CD11b-APC and DAPI as a viability dye prior to Imaging flow cytometry. The data were collected with the help of Scott Mordecai in the Department of Pathology flow and Image Cytometry Core (Amnis ImageStream, EMD Millipore).
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