Phenotypic characterization of canine BMSCs was performed as described.18 Adherent cells were dissociated with 0.05% trypsin‐ethylenediaminetetraacetic acid (Life Technologies) and resuspended in DMEM containing 10% fetal bovine serum. The cells were washed once with phosphate‐buffered saline (Life Technologies), and after centrifugation the phosphate‐buffered saline was removed and replaced with phosphate‐buffered saline containing 2% canine serum (AbD Serotec, Oxford, UK). The cells were placed on ice for 20 minutes, followed by incubation for a further 20 minutes on ice with monoclonal antibodies against cluster of differentiation 11b (CD11b; AbD Serotec), CD29‐PE (Abcam, Cambridge, UK), CD44‐APC (Biolegend, San Diego, CA), CD45‐eFlour (ebioscience, San Diego, CA), and CD90‐APC (ebioscience). The CD11b antibody was detected using goat polyclonal secondary antibody to mouse immunoglobulin G heavy and light chains (DyLight 488; Abcam). Isotype‐identical antibodies were used as controls. Flow‐cytometric analyses were performed using the Gallios system (Beckman Coulter, Danvers, MA). Propidium iodide (Sigma‐Aldrich, St. Louis, MO) was used to exclude dead cells from analyses. Each sample was assessed at least in triplicate. Data were analyzed using Kaluza software (Beckman Coulter).
Gallios system
Manufactured by Beckman Coulter
Sourced in United States
The Gallios system is a flow cytometer developed by Beckman Coulter. It is designed to analyze and sort cells based on their physical and fluorescent properties. The Gallios system offers high-performance capabilities for a wide range of applications in areas such as immunology, hematology, and cell biology.
Automatically generated - may contain errors
Lab products found in correlation
Facscan cytometer, by BD (2 mentions)
Trypsin ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Cd90 apc, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Bio-Rad (1 mentions)
Cd29 pe, by Abcam (1 mentions)
Cd44 apc, by BioLegend (1 mentions)
Dylight 488, by Abcam (1 mentions)
Cd11b, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Optilyse c, by Beckman Coulter (1 mentions)
5 protocols using gallios system
1
Phenotypic Characterization of Canine BMSCs
2
Flow Cytometry Analysis of Mouse Cells
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The antibodies used for flow cytometric analyses for mouse cells can be found in Supplementary Information . All cells were analyzed on a FACScan cytometer (BD Biosciences, Mountain View, CA) or a Gallios system (Beckman Coulter Life Sciences, Indianapolis, IN).
3
Flow Cytometry Analysis of Mouse Cells
4
Neutrophil Subset Analysis in STEMI
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To assess if STEMI patients showed increased circulating neutrophil subsets, flow cytometry analysis in two patients was performed. Blood was collected in EDTA-filled vacutainers and co-incubated with a mouse anti-human CD62L-ECD monoclonal antibody (Beckman Coulter, Brea, CA, USA) and a monoclonal CD16-APC-A700 antibody (Beckman Coulter, Brea, CA, USA) for 30 min. After incubation, blood was lysed for 10 min using Optilyse C (Beckman Coulter, Brea, CA, USA) followed by flow-cytometry measurements on the Gallios™ system (Beckman Coulter, Brea, CA, USA).
5
Immunoglobulin Binding and Phagocytosis of Mycobacterium tuberculosis
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To assess Mtb binding of immunoglobulins, 0.05 mL aliquots of BAL fluid obtained prior to and 8 weeks after vaccination, were incubated at 37°C for 1 hour with 107 CFU of Mtb H37Rv-dsRed. (Korbee et al., 2018) Subsequently, samples were equally divided over three vials and biotinylated detection antibodies specific for IgA, IgG or IgM (all from Mabtech) were added (final dilution 1:500). After 30 minutes of incubation at room temperature, streptavidin-FITC (final dilution 1:400) was added, and samples were incubated for a further 60 minutes toward detection of bound antibody. Samples were fixed overnight with 2% PFA before analysis.
For the phagocytosis assay, 5x105 THP1 cells were seeded in a 24-wells plate and activated by overnight incubation with 10ng/mL PMA at 37°C. The next day, 107 CFU of Mtb H37Rv-GFP was incubated with BAL fluid as described above, and subsequently added to the activated THP-1 cells. After 4 hours cells were dissociated with trypsin-EDTA and resuspended in 2% PFA for overnight fixation. The binding assay samples were subsequently acquired on a 4 laser, FACSAriaIII system; samples from the phagocytosis assay were acquired on a 3 laser, Beckman Coulter Gallios system. For both assays, Mtb H37Rv-dsRed or -GFP incubated in PBS only was taken along as a negative control.
For the phagocytosis assay, 5x105 THP1 cells were seeded in a 24-wells plate and activated by overnight incubation with 10ng/mL PMA at 37°C. The next day, 107 CFU of Mtb H37Rv-GFP was incubated with BAL fluid as described above, and subsequently added to the activated THP-1 cells. After 4 hours cells were dissociated with trypsin-EDTA and resuspended in 2% PFA for overnight fixation. The binding assay samples were subsequently acquired on a 4 laser, FACSAriaIII system; samples from the phagocytosis assay were acquired on a 3 laser, Beckman Coulter Gallios system. For both assays, Mtb H37Rv-dsRed or -GFP incubated in PBS only was taken along as a negative control.
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