The phagocytic and endocytic properties of the established cell line were evaluated using 2% pHrodo™ E. coli Bioparticles® (Life Technologies, Carlsbad, CA). Using a 24-well plate, 100,000 cells were plated per well and left overnight. Culture medium was removed and replaced by 2% pHrodo™ E. coli Bioparticles® diluted in Live Cell Imaging Solution (Life Technologies, Carlsbad, CA) for 1.5–2 h before imaging. Confocal images were obtained using Leica TCS SPE confocal system (Leica Microsystems, Buffalo Grove, IL) on excitation wavelength of 460 nm. Commercially available murine macrophage cell line J774.A (ATCC® TIB-67™), a canine HS cell line DH82, derived from a macrophage derived sarcoma, hemophagocytic HS (ATCC® CRL-10389™), and canine fibroblasts isolated from the tunica albuginea were used for functional comparison purposes.
Phrodo e coli bioparticles
Manufactured by Thermo Fisher Scientific
Sourced in United States
PHrodo E. coli bioparticles are fluorescent particles derived from E. coli bacteria. They are designed to be used in cellular assays to monitor phagocytosis and other cell-based activities.
Automatically generated - may contain errors
Lab products found in correlation
Live cell imaging solution, by Thermo Fisher Scientific (2 mentions)
Fluorescent microscope, by Olympus (1 mentions)
594 labeled secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
A14291dj, by Thermo Fisher Scientific (1 mentions)
Nucblue reagent, by Thermo Fisher Scientific (1 mentions)
Cd3 pacific blue clone sp34 2, by BD (1 mentions)
Cd8 apc h7 clone sk1, by BD (1 mentions)
Cd14 bv785 clone m5e2, by BioLegend (1 mentions)
Cd4 bv650 clone okt4, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Tcs spe confocal system, by Leica (1 mentions)
Crl 10389, by American Type Culture Collection (1 mentions)
Tib 67, by American Type Culture Collection (1 mentions)
Spectramax plate reader, by Molecular Devices (1 mentions)
Raw264.7 macrophage, by American Type Culture Collection (1 mentions)
Cd11b, by BD (1 mentions)
15 protocols using phrodo e coli bioparticles
1
Phagocytic and Endocytic Capacity of Cell Lines
2
Phagocytosis Assay with Microglial Cells
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Microglial cells were incubated with the pH-dependent pHrodo E. coli BioParticles (Thermo Scientific) for 1 h. After washing with PBS, cells were subjected to flow cytometry to measure the fluorescence intensity. Cells that were not incubated with microspheres served as negative controls and were used for background subtraction. Alternatively, after incubating with and removing unbound BioParticles, microglial cells were fixed in 4% paraformaldehyde, and stained with DAPI. The fluorescent signals were captured under a fluorescent microscope (Olympus) and analyzed using ImageJ.
3
Phagocytosis Assay for Microglia
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Primary microglia and HMC3 cells were plated into 12-well plates as described above. After 24 h, all cells were treated with myelin debris at 10 μg/mL. PHrodo E. coli bioparticles (P35366, Thermo Fisher, USA) were added to the cells for 2 h to evaluate the phagocytic ability. As negative controls, pre-treatment of Cytochalasin D was carried out for 30 min to prevent phagocytic uptake of cells. For fluorescence staining, cells were washed with PBS to remove non-phagocytosed pHrodo bioparticles followed by fixation with 4% PFA for 30 min. After blocked by 5% goat serum, cells were incubated with primary antibody rabbit anti-Iba1 (1:500, Wako, Japan) overnight at 4 °C. Cells were incubated with 594-labeled secondary antibodies (Invitrogen, USA) for 2 h and then mounted by Fluoromount-G with DAPI for 10 min. For flow cytometry assay, cells were trypsinized and centrifuged at 1000 rpm for 5 min. After that, cells were resuspended in FACS buffer to analyze the fluorescent intensity of FITC on a FACSVerse Flow Cytometer (BD Biosciences, USA).
4
Phagocytosis Assay of Sorted MDSCs
5
Phagocytosis of E. coli by THP-1 cells
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Differentiated Human THP-1 cells were incubated at 37 °C with pHrodo E. coli bioparticles (ThermoFisher Scientific, P35361) for 60 min as per the manufacturer’s instructions, followed by fixing the cell using 4% paraformaldehyde (PFA) and actin was stained using AlexaFlour-488 phalloidin. Wherever indicated, cytochalasin (CytoD) or SecinH3 were added to the THP-1 cells 30 min prior to incubating the cells with pHrodo E. coli bioparticles. The amount of phagocytosis was assessed by counting the internalized bacteria (red) in a minimum of 50 cells per condition.
6
Remote Injury Effects on Pneumonia
7
Phagocytosis of E. coli Bioparticles
8
Phagocytosis Assay of Neutrophils
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Neutrophils at the D7 PMN stage were pretreated with 20 nM RIST4721 (or DMSO) by adding drug to the culture media and incubating the cells for 30 min at 37 °C, 5% CO2. Next, 1 × 106 cells per sample were harvested by centrifugation at 250× g for 5 min, washed in PBS and resuspended in HBSS at 100 μL per sample. The drug and DMSO were added to the cell mixtures at the same concentration as used for the pretreatment step. The cells were then subjected to a phagocytosis assay as described previously [41 (link)]. Briefly, the cell/drug mixtures were transferred into 5 mL snap-cap tubes together with 710 μL of HBSS, 100 μL of mouse serum, 80 μL of NucBlue reagent and 10 μL of opsonized pHrodo E. coli bioparticles (Molecular Probes, Eugene, OR, USA). The tubes were secured with parafilm and incubated at 37 °C with gentle rocking for 1 h. The samples were then harvested through centrifugation at 1000× g for 5 min and resuspended in 35 μL HBSS with 2% FBS for processing with FlowSight imaging flow cytometer (Luminex).
9
Phagocytic Activity Measurement in Blood
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Phagocytic activity was measured on days 0, 15 and 30 using a commercial kit
(pHrodo E. coli BioParticles, Molecular Probes
Inc., Oregon, USA). Blood samples were collected by jugular puncture and placed
in heparinized tubes. Then, 100 μL of each sample was incubated with 20 μL of
pHrodo E. coli BioParticles, a reagent
provided by the commercial kit. For each blood sample two tubes were prepared
with the bioparticles; one was placed on ice, and the other kept in a water bath
at 37 ºC for 15 min. Next, the incubated samples were lysed, followed by
centrifugation and washing using the proper reagents as recommended by the
manufacturer. Two negative control samples were run together on each collection
day, both tubes with no bioparticles, but one placed on ice and the other kept
at 37 ºC. Samples were analyzed using a flow cytometer (FACSCanto, Becton
Dickinson Immunocytometry System, Mountain View, CA, USA), and the results were
expressed as the percentage of fluorescence signal inside the desired population
of neutrophils and monocytes. The target cell population was gated according to
its volume and complexity [36 (link)].
(pHrodo E. coli BioParticles, Molecular Probes
Inc., Oregon, USA). Blood samples were collected by jugular puncture and placed
in heparinized tubes. Then, 100 μL of each sample was incubated with 20 μL of
pHrodo E. coli BioParticles, a reagent
provided by the commercial kit. For each blood sample two tubes were prepared
with the bioparticles; one was placed on ice, and the other kept in a water bath
at 37 ºC for 15 min. Next, the incubated samples were lysed, followed by
centrifugation and washing using the proper reagents as recommended by the
manufacturer. Two negative control samples were run together on each collection
day, both tubes with no bioparticles, but one placed on ice and the other kept
at 37 ºC. Samples were analyzed using a flow cytometer (FACSCanto, Becton
Dickinson Immunocytometry System, Mountain View, CA, USA), and the results were
expressed as the percentage of fluorescence signal inside the desired population
of neutrophils and monocytes. The target cell population was gated according to
its volume and complexity [36 (link)].
10
Phagocytic Activity Assay Using pHrodo E. coli BioParticles
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Phagocytic activity was measured on days 1 and 28 using a commercial kit (pHrodo E. coli BioParticles, Molecular Probes Inc., Oregon, USA). The protocol consisted of incubation of 100 μL of a heparinized blood sample with 20 μL of pHrodo E. coli BioParticles reagent provided by the commercial kit (bioparticle:phagocyte ratio of 20:1). For each blood sample, two tubes were prepared with the bioparticles, with one tube placed on ice and the other kept at 37 °C in a water bath for 15 min. Then, the incubated samples were lysed, followed by centrifugation and washing using the proper reagents. Two negative control samples were analysed together on each collection day, both tubes with no bioparticles but one placed on ice and the other kept at 37 °C. Samples were analysed using flow cytometry (FACSCanto®, Becton Dickinson Immunocytometry System, Mountain View, CA, USA), and the results are shown as the percentage of fluorescence signal inside the desired population of phagocytosing neutrophils and monocytes.
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