Whole blood samples were obtained in heparin tubes. Blood samples were incubated with pHrodo E. coli (0.005 μg, Invitrogen) for 1 hour at 37°C or on ice. To each tube, Fc Block (15 μL, eBioscience) and cluster of differentiation (CD) 86 (5 μL, BioLegend) were added. CD86 was used to differentiate monocytes from other cell types, such as natural killer cells. Tubes were then incubated in the dark, on ice for 30 minutes with gentle agitation. The tubes were then treated with 1‐step Fix/Lyse Solution (eBioscience) and incubated for an additional 15 minutes at room temperature. Samples were then centrifuged at 500g for 5 minutes, washed with flow buffer, centrifuged again at 500g for 5 minutes, resuspended, and analyzed by flow cytometry (250 000 events per sample; BD FACSVerse, Becton, Dickinson and Company). Analysis was conducted using FlowJo software (FlowJo, LLC).
Phrodo e coli
Manufactured by Thermo Fisher Scientific
The PHrodo E.coli is a lab equipment product from Thermo Fisher Scientific. It is designed to measure the pH-dependent fluorescence of E. coli cells in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Fc block, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
1 step fix lyse solution, by Thermo Fisher Scientific (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
96 well tissue culture plate, by Corning (1 mentions)
Celltracker blue cmac, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Dura luminol substrate, by Thermo Fisher Scientific (1 mentions)
Fluorobrite dmem media, by Thermo Fisher Scientific (1 mentions)
Incucyte, by Sartorius (1 mentions)
24 well plate, by Corning (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
E coli k12 ultra pure lps, by InvivoGen (1 mentions)
9 protocols using phrodo e coli
1
Monocyte Phagocytosis Assay
2
Phagocytosis Assay for Hemocytes
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The assay for phagocytosis was performed according to the method described by Cuttell et al., (2008) (link), with minor modifications. After isolation of hemocytes, they were prepared in a 96-well tissue culture plate (CoStar, Washington). Cells were incubated for 1 hr counterstained with 20 μM Cell Tracker Blue CMAC (Molecular Probes) (Life Technologies, Carlsbad, CA) and then washed in PBS. 5 μl of the drug solution were mixed with 45 μl of Grace’s medium were added into each well for 30 min. Bacterial phagocytosis assays using pHrodo E. coli (Invitrogen, Carlsbad, CA) were performed according to the manufacturer’s instructions. The proportion of cells that had phagocytosed labeled E. coli was determined under a florescence microscope (Nikon Eclipse TS100, Nikon, Japan) at 200 × in five different fields.
3
Phagocytosis and Superoxide Assays
4
Assessing Macrophage Phagocytic Function
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Chronic L. sigmodontis-infected mice and uninfected controls were i.p. injected with 100µg of pHrodo-E. coli or S. aureus BioParticles from Thermo Scientific. At 90 min post injection (E. coli) or 3h post injection (S. aureus), mice were euthanized and peritoneal lavage was analyzed by flow cytometry to assess the frequencies of pHrodo positive macrophages.
5
Quantifying Microglia Phagocytosis via pH-rodo E. coli
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Experimental and Imaging Set-up: Primary microglia were prepared as described above. Microglia were seeded at 30,000 cells / well in a 96-well plate. After 24 hours, media was replaced with FluoroBrite DMEM media (ThermoFisher) with 10% FBS and 0.015 mg pH-rodo E. coli (ThermoFisher; P35366) was added to each well. 4 fields of view were taken for each well every 30 minutes using Incucyte (Essen BioScience). Background fluorescence was subtracted and population-level fluorescence information was determined through the Incucyte analysis software using Top-Hat and threshold based on negative control well with no pH-rodo E. coli.
6
Phagocytosis Assay with pHrodo E.coli
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For assessment of phagocytosis, pHrodo E.coli (#P35361 Life Technologies) were thawn freshly on ice and separated into single cells using ultrasound technology. After initial maturation of monocytes/macrophages in differentiation medium II for 5 days, cells were harvested and 2 × 106 cells /500 µl RPMI (without phenolred and with 20 mmol/l Hepes) were incubated for 2 h with 25 µl pHrodo E.coli either at 4 °C or 37 °C, followed by incubation on ice for 10 min, before flow cytometric analysis.
7
Mouse Mindin Protein Analysis
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Mouse recombinant mindin protein was purchased from R&D Systems (Minneapolis, MN). The following antibodies were used: rabbit monoclonal anti‐GAPDH and CD11b antibodies [cat no: 20991‐1‐AP] (Proteintech, Rosemont, Pennsylvania); mouse anti‐Myc‐tagged antibody (Proteintech); rabbit monoclonal anti‐phospho‐MEK1/2, anti‐MEK1/2, anti‐phospho‐Zap70 and anti‐NF‐κB p65 antibodies (Cell Signalling, Beverly, MA); rabbit monoclonal anti‐phospho‐SykY323, anti‐phospho‐SykY525/526, anti‐Syk, anti‐CD11b [clone no: M1/70] and anti‐CD11b [cat no:ab128797] antibodies (Abcam, Cambridge, MA) and mouse monoclonal anti‐CD18 [clone no: M18/2] antibody (Abcam); mouse monoclonal anti‐CD18 antibody [cat no: CTB104], rabbit and mouse IgG antibodies and anti‐HA antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); fluorescence isothiocyanate (FITC)‐labelled goat anti‐rabbit secondary antibody and tetramethyl rhodamine isothiocyanate (TRITC)‐labelled goat anti‐mouse secondary antibody (Cell Signalling, Beverly, MA); mouse polyclonal anti‐PARP antibody (BioSource, Camarillo, CA); rabbit monoclonal anti‐phospho‐ERK1/2 antibody and ERK1/2 antibody (Promega, Madison, WI);pHrodo E.coli (Life Technologies, Grand Island, NY);Phalloidin (Solarbio ShangHai China).
8
Phagocytosis Assay using iPSC-MGLCs
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iPSC-MGLCs were seeded at 1 × 105 cells/well in 24-well plates (Corning) 48 hr before the assay. As a negative control, cells were preincubated for 30 min with 10 μM cytochalasin D (Sigma). Cells were incubated with pHrodo E. coli (50 μg) or pHrodo zymosan (25 μg) (Life Technologies) particles for 2 hr. Cells were analyzed with a Becton Dickinson FACSCalibur flow cytometer, and results were analyzed with Flowing software (Cell Imaging Core of the Turku Centre for Biotechnology, flowingsoftware.btk.fi ).
9
Autophagy Induction in Macrophages
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