The 5-ethynyl-2ʹ-deoxyuridine (EdU) used in the in vitro and in vivo proliferation assays was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). RPMI1640 was purchased from Nacalai Tesque. Lipid A purified from Salmonella minnesota (Re-595) was purchased from Sigma–Aldrich (Merck, Darmstadt, Germany). Brefeldin A was purchased from BioLegend (San Diego, CA, USA). pHrodo Green E. coli bioparticles Conjugate for Phagocytosis was purchased from Invitrogen.
Phrodo green e coli bioparticles conjugate for phagocytosis
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PHrodo Green E. coli BioParticles Conjugate is a fluorescent probe designed for the detection and measurement of phagocytosis. It consists of pH-sensitive pHrodo dye that is conjugated to E. coli-derived particles. The fluorescence of the probe increases upon internalization into acidic environments, such as phagosomes, allowing for the visualization and quantification of phagocytic activity.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 magnetic beads, by Miltenyi Biotec (2 mentions)
Anti cd28, by Thermo Fisher Scientific (2 mentions)
Anti cd3, by Thermo Fisher Scientific (2 mentions)
Re 595, by Merck Group (1 mentions)
Rpmi 1640, by Nacalai Tesque (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Fluorescence microplate reader, by PerkinElmer (1 mentions)
Facsaria 3 sorter, by BD (1 mentions)
P35361, by Thermo Fisher Scientific (1 mentions)
Phrodo red e coli bioparticles conjugate for phagocytosis, by Thermo Fisher Scientific (1 mentions)
Mouse macrophage colony stimulating factor m csf, by BioLegend (1 mentions)
Human quantikine elisa kit, by R&D Systems (1 mentions)
Nadp nadph assay, by Abcam (1 mentions)
8 protocols using phrodo green e coli bioparticles conjugate for phagocytosis
1
Proliferation Assays with EdU
2
Quantifying Phagocytic Activity of Macrophages
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The phagocytic capability of alveolar macrophages and blood monocytes was tested with pH-sensitive pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (Invitrogen, Waltham, MA, United States), according to the manufacturer’s instructions. The conjugates are nonfluorescent at neutral pH, but fluoresce bright green at acidic pH, such as in phagosomes. Mononuclear cells collected from BALF or blood were seeded onto a tissue culture dish and incubated in RPMI 1640 10% fetal bovine serum (FBS) with 1% penicillin/streptomycin for 2 h at 37°C, 5% CO2. Cells that adhere to the plate are macrophages (26 (link), 27 (link), 34 (link)). Briefly, a total of 105 alveolar macrophages were plated on a 96-well plate. Cells were washed with saline (0.9% NaCl) and incubated with fluorescent pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (0.5 mg/mL) for 2 h. Phagocytosis was quantified by measuring intracellular fluorescence emitted by engulfed particles at 585 nm in a microplate fluorescence reader (PerkinElmer, Waltham, MA, United States).
3
Phagocytosis Assay in Brain Slices and Cell Suspensions
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For the phagocytosis assay in the acute brain slice culture, brain slices were prepared as previously described and incubated with pHrodo Red E. coli BioParticles Conjugate for Phagocytosis (0.25 mg/ml; P35361, Invitrogen) or pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (P35366, Invitrogen) in the Leibovitz’s L-15 medium (11415064, Gibco) containing 10% FBS.
For the phagocytosis assay in cell suspensions, microglia suspensions were prepared as previously described and incubated with pHrodo Red E. coli BioParticles Conjugate for Phagocytosis (0.8 mg/ml; P35361, Invitrogen) in the Leibovitz’s L-15 medium (11415064, Gibco) containing 10% FBS for 1 hour at 30°C. After incubation, cell suspensions were analyzed in a FACSAria III sorter (BD Biosciences).
For the phagocytosis assay in cell suspensions, microglia suspensions were prepared as previously described and incubated with pHrodo Red E. coli BioParticles Conjugate for Phagocytosis (0.8 mg/ml; P35361, Invitrogen) in the Leibovitz’s L-15 medium (11415064, Gibco) containing 10% FBS for 1 hour at 30°C. After incubation, cell suspensions were analyzed in a FACSAria III sorter (BD Biosciences).
4
Phagocytosis Assay of NMR and NPM1 Cells
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NMR cells were used after 8 days of culture with 20 ng/ml mouse macrophage colony stimulating factor (M-CSF) (BioLegend) or immediately after cell sorting or one day of culture (NPM1 cells). Cells were cultured with pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (Thermo Fisher Scientific) for 2 hours. Cultured cells were washed, fixed with 4% paraformaldehyde, and stained with the CD11b antibody and DAPI. Sorted cells and NPM1 cells were stained with 5 μg/ml Hoechst 33342 without fixation. Stained cells were observed by fluorescence microscopy. Image analyses were performed using ImageJ.
5
Assessing Cellular Metabolism and Immunity
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Cell viability was assessed using Trypan blue and counted using a hemocytometer. Intracellular reduced and oxidized glutathione levels were measured and normalized per 1 million cells as previously described (33 (link)). NADP and NADPH levels were measured using an NADP/NADPH Assay (Abcam) and normalized by cell count. Nitric oxide production was assessed by a Griess assay on the culture supernatant (34 (link)). Phagocytic capacity of monocytes was assessed by exposing the monocytes to the pHrodo Green E. coli BioParticles Conjugate for Phagocytosis (ThermoFisher Scientific) for 30 minutes and the endpoint signal was measured using a spectrophotometer at OD 495nm. IL-6, IL-8, and IL10 levels were measured in culture supernatant using Quantikine Human ELISA kits (R&D Systems).
6
Robust Immune Cell Isolation and Characterization
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The function and cell identity of AM, MDM, and CD4+ T cells was determined by morphology, marker staining, functional assays, and RT-qPCR (see above). The purity of AM cells was analyzed by marker staining using anti-CD68 antibody ([EPR20545] (ab213363), abcam) and pHrodo™ Green E. coli BioParticles™ Conjugate for Phagocytosis (P35366, Thermofisher). Using this method, we obtained AMs with a purity of > 95.8% and are functional of > 91.6% with neglectable T cell contamination (0.9% for AMs and 1.6% for MDMs).
7
Phagocytosis and T Cell Differentiation
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The phagocytosis assay of macrophages engulfing pHrodo-labeled bacteria was performed by pHrodo Green E. coli BioParticles conjugate for phagocytosis (Thermo Fisher Scientific, catalog P35366) according to instructions. In brief, pHrodo Green E. coli BioParticles conjugate was added to the cell culture supernatant, which is engulfed by macrophages. Flow cytometry was used to detect the fluorescence level of macrophage. The experimental procedure for detecting the ability of macrophages to regulate T cell differentiation is as follows: the CD4+ T cells in spleen from the C57BL/6 mice were sorted by CD4 magnetic beads (Miltenyi Biotec, catalog 130-117-043) and stimulated with 2.5 μg/mL anti-CD3 (eBioscience, catalog 16-0031-82) and 3 μg/mL anti-CD28 (eBioscience, catalog 16-0281-82) for 48 hours; the activated CD4+ cells were coincubated with different macrophage culture supernatants, and the differentiation ratio of CD4+ T cells was measured 24 hours later.
8
Phagocytosis and T Cell Differentiation
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The phagocytosis assay of macrophages engulfing pHrodo-labeled bacteria was performed by pHrodo Green E. coli BioParticles conjugate for phagocytosis (Thermo Fisher Scientific, catalog P35366) according to instructions. In brief, pHrodo Green E. coli BioParticles conjugate was added to the cell culture supernatant, which is engulfed by macrophages. Flow cytometry was used to detect the fluorescence level of macrophage. The experimental procedure for detecting the ability of macrophages to regulate T cell differentiation is as follows: the CD4+ T cells in spleen from the C57BL/6 mice were sorted by CD4 magnetic beads (Miltenyi Biotec, catalog 130-117-043) and stimulated with 2.5 μg/mL anti-CD3 (eBioscience, catalog 16-0031-82) and 3 μg/mL anti-CD28 (eBioscience, catalog 16-0281-82) for 48 hours; the activated CD4+ cells were coincubated with different macrophage culture supernatants, and the differentiation ratio of CD4+ T cells was measured 24 hours later.
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