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Phrodo red e coli bioparticles conjugate for phagocytosis

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The PHrodo Red E.coli BioParticles conjugate is a fluorescent reagent designed to study phagocytosis. It consists of pH-sensitive dye-labeled E. coli particles that increase in fluorescence upon internalization into acidic phagocytic vesicles.

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Lab products found in correlation

Cytochalasin d, by Merck Group (3 mentions) Lsrii fortessa, by BD (1 mentions) Facsaria 3 sorter, by BD (1 mentions) P35361, by Thermo Fisher Scientific (1 mentions) Phrodo green e coli bioparticles conjugate for phagocytosis, by Thermo Fisher Scientific (1 mentions) Eclipse te2000 inverted microscope, by Nikon (1 mentions) Src inhibitor pp2, by Merck Group (1 mentions) Recombinant human gm csf, by Thermo Fisher Scientific (1 mentions) Collagen type 1 from rat tail, by Merck Group (1 mentions) M csf, by Thermo Fisher Scientific (1 mentions) Formaldehyde, by Merck Group (1 mentions) Lsrii flow cytometer, by BD (1 mentions)

11 protocols using phrodo red e coli bioparticles conjugate for phagocytosis

1

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).
Wu S., Nguyen L.T., Pan H., Hassan S., Dai Y., Xu J, & Wen Z. (2020). Two phenotypically and functionally distinct microglial populations in adult zebrafish. Science Advances, 6(47), eabd1160.
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2

Phagocytosis Assay for Dendritic Cells

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DC2.4WT or DC2.4Dab2−/− cells (105) were incubated for 1 h with or without 10 μM cytochalasin D (Sigma Aldrich) in complete media. pHrodo Red E. coli Bioparticles Conjugate for Phagocytosis were added to cells according to the manufacturer instructions (Invitrogen, Carlsbad, CA) and incubated for an additional 1 h at 37°C. Phagocytosis was also assessed in DC2.4WT or DC2.4Dab2−/− cells after incubation of 105 cells with IgG-coated FluoSpheres carboxylate (2.0 μm, red 580/605) (Life Technologies) at 1:25 ratio for 1 h in complete DMEM. Cells were washed with PBS prior to acquisition using an LSRII Fortessa (BD Biosciences). The data was analyzed for the median fluorescence intensity (MFI) and the percentage of phagocytic cells using FlowJo (Tree Star).
Figliuolo da Paz V., Jamwal D.R., Gurney M., Midura-Kiela M., Harrison C.A., Cox C., Wilson J.M., Ghishan F.K, & Kiela P.R. (2019). Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells. Frontiers in Immunology, 10, 304.
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3

Quantifying Phagocytosis via Bioparticle Uptake

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Uptake of bioparticles (pHrodo Red E. coli Bioparticles Conjugate for Phagocytosis; Molecular Probes; Eugene, OR) was used to quantify phagocytosis in vitro by the appearance of fluorescence when the bioparticle is incorporated into the acidic environment of the phagolysosome. Cells (1 X 105 /well) were incubated (37°C, 5% CO2) in a 96 well plate for an hour. Adherent cells were washed with PBS, and exposed to 200 μl of either fibrocyte medium or bioparticles prepared per manufacturer’s instructions in fibrocyte media. After two hours of incubation, cells were washed with PBS, fixed and assessed for bioparticle expression by flow cytometry. For a negative control, cytochalasin D (5 μg/mL, Sigma Aldrich) was added to fibrocyte media for 45 minutes which temporarily binds actin filaments to inhibit phagocytosis.
Collins D., Fry C., Moore B.B, & Nemzek J.A. (2019). Phagocytosis by Fibrocytes as a Mechanism to Decrease Bacterial Burden and Increase Survival in Sepsis. Shock (Augusta, Ga.), 51(4), 464-471.
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4

Phagocytic Activity Quantification in Flies

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Phagocytic activity was assessed by measuring fluorescence following the injection of pHrodo Red E. coli BioParticles Conjugate for Phagocytosis (Molecular Probes). A 4 mg/mL suspension of pHrodo particles was diluted 1:4 in ES products such that each co-injection contained 310 larval equivalents in a 1 mg/mL solution of pHrodo particles. Upon injection, flies were incubated at 25°C for 1 h at which time the dorsal side of the abdomen associated with the pericardial nephrocytes was imaged using a Nikon ECLIPSE Ni microscope at 10x magnification with a Zyla (ANDOR) 5.5 camera. Corrected total fluorescence was measured using ImageJ software.
Kenney E., Hawdon J.M., O'Halloran D, & Eleftherianos I. (2019). Heterorhabditis bacteriophora Excreted-Secreted Products Enable Infection by Photorhabdus luminescens Through Suppression of the Imd Pathway. Frontiers in Immunology, 10, 2372.
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5

Quantifying Phagocytosis of E. coli Bioparticles

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pHrodo Red E.coli BioParticles conjugate for Phagocytosis were purchased from ThermoFisher, USA and reconstituted to 1 mg/ml in 10% FBS-containing media. Reconstituted Bioparticles were added at a final concentration of 0.1 mg/ml to IDUA-HSPC-derived macrophages and incubated at 37 °C for 1 h. The cells were then washed and bathed in imaging media (DMEM Fluorobright, 15 mM HEPES, 5% FBS). Imaging followed using the appropriate absorption and fluorescence emission maxima (560 and 585 nm, respectively) with a BZ-X710 Keyence fluorescence microscope. Images were quantified using ImageJ 1.51.
Scharenberg S.G., Poletto E., Lucot K.L., Colella P., Sheikali A., Montine T.J., Porteus M.H, & Gomez-Ospina N. (2020). Engineering monocyte/macrophage−specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing. Nature Communications, 11, 3327.
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6

THP-1 Phagocytosis Assay

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The cells were treated in the same manner as for the polarisation assay, but this time 10.000 THP-1 were seeded in 24-well plates. On day 5, the medium was replaced with HBSS medium supplemented with 2% FBS and 50 ug of pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis (Thermo Fisher) was added to each well. Fluorescence intensity was measured for 4 h taking pictures every 5 min with an Eclipse TE2000 Inverted Microscope (Nikon).
García-Vílchez R., Añazco-Guenkova A.M., Dietmann S., López J., Morón-Calvente V., D’Ambrosi S., Nombela P., Zamacola K., Mendizabal I., García-Longarte S., Zabala-Letona A., Astobiza I., Fernández S., Paniagua A., Miguel-López B., Marchand V., Alonso-López D., Merkel A., García-Tuñón I., Ugalde-Olano A., Loizaga-Iriarte A., Lacasa-Viscasillas I., Unda M., Azkargorta M., Elortza F., Bárcena L., Gonzalez-Lopez M., Aransay A.M., Di Domenico T., Sánchez-Martín M.A., De Las Rivas J., Guil S., Motorin Y., Helm M., Pandolfi P.P., Carracedo A, & Blanco S. (2023). METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer. Molecular Cancer, 22, 119.
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7

Phagocytosis of E. coli Bioparticles

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pHrodo Red E. coli BioParticles conjugate for Phagocytosis were purchased from ThermoFisher, USA and reconstituted to 1 mg/mL in 10% FBS-containing media. Reconstituted Bioparticles were added to IDUA-HSPC-derived macrophages and incubated at 37 °C for one hour. The cells were then washed and bathed in imaging media (DMEM Fluorobright, 15 mM HEPES, 5% FBS). Imaging followed using the appropriate absorption and fluorescence emission maxima (560 nm and 585 nm, respectively).
Gomez-Ospina N., Scharenberg S.G., Mostrel N., Bak R.O., Mantri S., Quadros R.M., Gurumurthy C.B., Lee C., Bao G., Suarez C.J., Khan S., Sawamoto K., Tomatsu S., Raj N., Attardi L.D., Aurelian L, & Porteus M.H. (2019). Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I. Nature Communications, 10, 4045.
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8

Phagocytic Activity of BV2 Microglia

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Phagocytic activity of BV2 microglia was detected using engulfment of pHrodo Red E. coli BioParticles™ Conjugate for Phagocytosis (ThermoFisher Scientific, #P35361). Cells were treated in last hour of incubation with 1:1000 dilution of pHrodo red E. coli BioParticles™. Cells were washed with FACS buffer and resuspended in 1% PFA for 1 hour at room temperature. The cells were washed twice in FACS buffer and run on the CytoFLEX Flow Cytometer. Flow cytometry data was analysed using gating for cell size, granularity, singlet cell population, and phycoerythrin (PE) red-channel signal to detect cells with bead engulfment. FlowJo was used to assess the percent of phagocytic positive cells gated on the no stain in the PE channel and the median fluorescent intensity (MFI) of the positive population.
Meleady L., Towriss M., Kim J., Bacarac V., Dang V., Rowland M.E, & Ciernia A.V. (2023). Histone deacetylase 3 regulates microglial function through histone deacetylation. Epigenetics, 18(1), 2241008.
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9

Cell Culture Reagents and Neutrophil Assays

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All cell culture reagents unless specified elsewhere, were from Life Technologies, (Carlsbad, CA, USA). All chemicals not specified are from Sigma-Aldrich (St. Louis, MO, USA). Human neutrophil elastase (NE) was from Athens Research & Technology (Athens, GA, USA). Collagen Type I from rat tail and Src inhibitor PP2 were obtained from EMD Millipore (Burlington, MA, USA). Human recombinant GM-CSF and M-CSF were from Peprotech (Rocky Hill, NJ, USA). pHrodo Red E. coli BioParticles Conjugate for phagocytosis was from Thermo Fisher Scientific.
Krotova K., Khodayari N., Oshins R., Aslanidi G, & Brantly M.L. (2020). Neutrophil elastase promotes macrophage cell adhesion and cytokine production through the integrin-Src kinases pathway. Scientific Reports, 10, 15874.
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10

Phagocytosis Assay of E. coli

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Whole blood from healthy donors and patients was treated according to the
respective protocol, as shown below (phagocytosis experiments). Here,
the second hit was not performed with purified LPS, but with
fluorescently labelled E. coli cells. After
pre-treatment with LPS (first hit), the isolated PBMCs from healthy
donors were harvested, washed and incubated with non-opsonised
FITC-labelled E. coli (second hit). Whole blood from
patients was incubated directly with non-opsonised FITC-labelled
E. coli cells (PHAGOTEST®; ORPEGEN Pharma,
Heidelberg, Germany) or with endocytosis/acidification-sensitive
pHrodo-conjugated E. coli bioparticles (pHrodo™ Red
E. coli BioParticles™ Conjugate for
Phagocytosis; Life Technologies, Carlsbad, CA).29 (link) The percentage of phagocytic monocytes and phagocytic activity
per cell were analysed by flow cytometry as recommended by the
manufacturer.
Duerr C., Bacher A., de Martin A., Sachet M., Sadeghi K., Baumann S., Heinz C, & Spittler A. (2019). The novel polyclonal Ab preparation trimodulin attenuates ex vivo endotoxin-induced immune reactions in early hyperinflammation. Innate Immunity, 25(6), 374-388.
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