To assess phagocytic activity, the number of phagocytic cells and the mean fluorescence intensity (MIF) of pHrodo Green E. coli (Fisher Sci., Waltham, MA, USA) in phagocytic cells were determined. After 72 h of co-incubation with DCPD, OCP, and HAp, cells were incubated with 1 mg/mL pHrodo Green E. coli (Fisher Sci., Waltham, MA, USA) for 2 h after cell incubation in a CO2 incubator. To control for nonspecific staining, cells were preincubated with 10 μg/mL cytochalasin D (MerckMillipore, St. Louis, MO, USA). Measurements were performed using a BD Accuri C6 Cell Analyzer (BD/Fisher Sci., Franklin Lakes, NJ, USA) [33 (link)].
Phrodo green e coli
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PHrodo Green E. coli is a lab equipment product designed to measure the pH of E. coli samples. It uses a pH-sensitive fluorescent dye to indicate changes in the pH of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Daf fm da, by Thermo Fisher Scientific (3 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Id7000, by Sony (1 mentions)
Az100m, by Nikon (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Phrodo red s aureus, by Thermo Fisher Scientific (1 mentions)
Femtojet, by Eppendorf (1 mentions)
Nanoject 2, by Drummond (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Murine m csf, by R&D Systems (1 mentions)
Phrodo red s aureus bioparticles, by Thermo Fisher Scientific (1 mentions)
Hepes, by AppliChem (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hypoxia green reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Jsh 23, by Thermo Fisher Scientific (1 mentions)
11 protocols using phrodo green e coli
1
Assessing Phagocytic Activity by Flow Cytometry
2
Flow Cytometric Analysis of pHrodo E. coli Uptake
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Spleen cells were incubated with 50 µg/mL pHrodo Green E. coli (Invitrogen) dissolved in RPMI medium for 1 h at 37°C. After blocking with anti-CD16/32 (clone 95) mAb, the samples were stained by fluorescent dye-conjugated mAbs. pHrodo Green positive cells were detected using a spectral flow cytometer ID7000 (Sony Biotechnology).
3
Phagocytosis Assay in Drosophila
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To assay E. coli and S. aureus phagocytosis in adults, approximately eight to ten flies with an equal distribution of females and males per genotype were injected with ~0.2 μl of 1 mg/mL pHrodo green E. coli or pHrodo red S. aureus (Invitrogen, Carlsbad, CA) using a FemtoJet microinjection system (Eppendorf). They were then incubated for 1 hr at room temperature. Fluorescently labeled particles were visualized through the cuticle using a florescence microscope (Nikon AZ100M, Nikon, Japan). We use Image J software to quantify the results. Relative fluorescence calculated as: [fluorescence]dorsal vein area/[fluorescence]adjacent area.
To assay phagocytosis of beads, flies were first injected with approximately 36.8 nl PBS or 1 μg/μl 5-HT dissolved in PBS using a Drummond Scientific Nanoject II. Flies were incubated at room temperature for 30 min and then injected with approximately 27 nl of 1.0 μm Red Fluorescent Carboxylate Modified FluoSpheres diluted 1: 2 (Invitrogen), incubated at room temperature for 10 min, injected with 36.8 nl 0.4% Trypan blue (Invitrogen), and then mounted and visualized as described above.
To assay phagocytosis of beads, flies were first injected with approximately 36.8 nl PBS or 1 μg/μl 5-HT dissolved in PBS using a Drummond Scientific Nanoject II. Flies were incubated at room temperature for 30 min and then injected with approximately 27 nl of 1.0 μm Red Fluorescent Carboxylate Modified FluoSpheres diluted 1: 2 (Invitrogen), incubated at room temperature for 10 min, injected with 36.8 nl 0.4% Trypan blue (Invitrogen), and then mounted and visualized as described above.
4
In vivo and in vitro phagocytosis of E. coli
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Mice received an injection of 100 µg pHrodo™ green-E. coli (Thermo Fisher Scientific) in the presence or absence of 10 µg/g BW L-polyphosphates. Small volumes of blood were sampled in EDTA-tubes from the retro-orbital sinus of anaesthetized mice at several time points. Mice were sacrificed after 4−24 h and peritoneal cells were collected as described above and processed on ice. Following flow cytometry surface marker staining, live cells were analyzed using a FACSCanto II (Becton Dickinson) as described above. To control for auto-fluorescence, additional mice were challenged with fixed E. coli without pHrodo™ conjugate and used to obtain fluorescence minus one (FMO) controls. To determine the amounts of total undigested pHrodo™ green-E. coli bioparticles, 200 µl of peritoneal lavage was centrifuged 5 min at 10,000 × g, the pellet was re-suspended and incubated for 10 min in 2 N H2SO4 and pHrodo™ green fluorescence was determined with a Fluoroskan Ascent FL (Thermo Fisher Scientific). For the in vitro phagocytosis assay, BMDM were challenged with 330 µg/ml pHrodo™ green-E. coli in the presence or absence of 50 µM S- or L-polyphosphates. The fluorescence signals were acquired with a Fluoroskan Ascent FL at 4 h after stimulation.
5
Quantifying Phagocytosis of Bacterial Particles
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For assessment of phagocytosis, 1 × 105 cells were seeded in 12-well plates in standard medium. After >12 hours of settling, cells were incubated for 2 hours with pHRodo Green E. coli or pHRodo Red S. aureus BioParticles (Thermo Fisher Scientific) at a concentration of 1:20. Phagocytosis was performed in phenol-free RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FCS, 2% HEPES (AppliChem, Darmstadt, Germany), 1% L-glutamine (Invitrogen, Darmstadt, Germany) and 10 pg/mL murine M-CSF (R&D Systems, Minneapolis, MN, USA). Subsequently, cells were washed extensively, and the amount of incorporated BioParticles was evaluated by flow cytometry.
6
Comprehensive Reagent Reference for Cell Analysis
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Hypoxia green reagent; pHrodo Green E. coli; DAF-FM DA; JSH-23; QNZ; ABT-263; and ABT-737 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The fetal bovine serum was from Gibco (Gibco, Sigma Aldrich Company Ltd., Waltham, MA, USA). Intracellular staining permeabilization wash buffer; Human TruStain FcX (Fc Receptor Blocking Solution); PE anti-human Ki-67; isotype control antibodies PE Mouse IgG1 k isotype Ctrl; APC Mouse IgG1 k isotype Ctrl; FITC Mouse IgG1 k isotype Ctrl; PE Mouse IgG2a k isotype; PE Mouse IgG2b k isotype and antibodies APC anti-human CD11b; PE anti-human CD284; PE anti-human CD36; PE anti-human CD33; PE anti-human HLA-DR; PE anti-human HIF-1α were from BioLegend (San Diego, CA, USA). MycoFluor™ Mycoplasma Detection Kit was from Molecular Probes Inc. (Eugene, Oregon, USA). FITC anti-human CD68 was from BD Bioscience (Franklin Lakes, NJ, USA). Culture medium RPMI 1640; 2-mercaptoethanol; PBS; Calcein AM; Bisbenzimide Hoechst 33342 (H33342); propidium iodide (PI), resazurin; LPS of E. coli O111: B4; antibodies FITC anti-human CD11c; FITC anti-human CD14; FITC anti-human CD54; NF-kB activation inhibitor IV, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Quantifying Phagocytosis of Fluorescent E. coli
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Phagocytosis of fluorogenic Escherichia coli particles (pHrodo green E coli, Thermo Fisher Scientific) was analyzed using HEK293T ADAM10/17 -/-, PMA-induced THP-1, and U937 cells as well as primary macrophages (BMDMs) from mice. Cells were seeded in 96-well plates at a density of 8 × 10 4 cells per well 24 hours before phagocytosis measurements. After collection of the supernatants cells were washed two times with Live cell imaging solution (Thermo Fisher Scientific). E coli particles were added in a suspension of 1 μg/mL in Live cell imaging solution at a total of 50 μL/well. As a negative control, phagocytosis was inhibited with 10 µM of cytochalasin D (Life technologies), which was added 30 minutes prior to addition of pHrodo E coli bioparticles. Samples were incubated at 37°C for 120 minutes. Afterward, the suspension was carefully collected and cells were washed three times with MACS buffer (PBS pH 7.4, 0.5% (w/v) of BSA (Albumin Fraction V), 2 mM of EDTA). Cells were subsequently analyzed by either fluorescence-activated cell scanning (FACS) or fluorescence measurements with a microplate reader (Spark®, Tecan Group Ltd.). For Flow cytometric analysis of the phagocytosis assay, we used the FACSCanto (BD Biosciences). Data were analyzed with FACSDiva (BD Biosciences) and FLOWJO Software (Tree Star Inc).
| 6679 BERNER Et al.
| 6679 BERNER Et al.
8
Neutrophil Phagocytosis Assay with pHrodo E. coli
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Isolated human neutrophils (1.5 × 105 cells/100 μL) were challenged with 333 μg/mL pHrodo green E. coli (Thermo Fisher Scientific) in the presence or absence of polyphosphates at 37°C in the Fluoraskan Ascent FL fluorometer (Thermo Fisher Scientific). The fluorescence signals were acquired every 5 min for 7 h.
9
Phagocytic Capacity of Neutrophils in Psoriasis
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To assess the phagocytic capacity, neutrophils from healthy controls and psoriasis patients were isolated and incubated with pHrodo Green E coli (2 mg/mL; P35366, Thermo Fisher Scientific, USA) at 37 °C for 30 min. Incubation was stopped by the addition of 2 mL of PBS, and cells were then washed 3 times. Cells were divided into two parts, the first of which was used for flow staining. After incubated with PE-Cy7 conjugated anti-human CD15 (301924, 1:100, BioLegend) and PE conjugated anti-human CXCR4 (306506, 1:100, BioLegend) at 4 °C for 30 min. Phagocytic uptake was analyzed by flow cytometry and expressed as median fluorescence intensity (MFI). The remaining cells, which were not used for flow staining, were incubated with Hoechst 33258 (C0021, 1:1000, Solarbio technology) for 15 min for following confocal microscope (LSM880, Carl Zeiss, Germany).
10
Immunological Assays and Reagents Database
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pHrodo Green E. coli, PE anti-mouse/human Ki-67 antibodies, PE Rat IgG2b k Isotype Ctrl, and IgE Mouse ELISA Kit were purchased from Thermo Scientific (Waltham, MA, USA). Span® 85 (S7135), TWEEN® 80 (S7135), Deuterium oxide (151882), Culture media DMEM and RPMI 1640, L-glutamine, Pokeweed Mitogen (PWM), phytohemagglutinin (PHA), cytochalasin D, Methyl Methanesulfonate, Saponin, ELISA kits Mouse IL-2 ELISA Kit, TWEEN® 20, Human Serum Albumin, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Turpentine Oil (State Standard 1571-82, Top Grade RPC 24 1611 0120) was purchased from «IB Smirnova» (Moscow, Russia). Drabkin Method Kit was purchased from Agat-Med Ltd. (Moscow, Russia). EasySep™ Mouse Monocyte Isolation Kit was purchased from StemCell Technologies Inc. (Vancouver, BC, Canada). Mouse IFN-gamma Quantikine ELISA Kit was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Mouse TNF alpha ELISA Kit, Mouse IgM ELISA Kit, and Abcam Histamine ELISA kit were purchased from Abcam (Cambridge, UK). Mouse IgG ELISA Kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). All other reagents used were of analytical reagent quality.
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