Phrodo green

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PHrodo green is a fluorogenic pH indicator dye used for measuring pH changes in various biological applications. It exhibits increased fluorescence at acidic pH levels, making it a useful tool for monitoring pH-dependent processes in live cells and other systems.

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24 protocols using phrodo green

Quantitative Analysis of Phagocytosis

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The quantitative analysis of phagocytosis was performed by measuring the uptake of CFSE-labeled E. coli particles by flow cytometry. The fluorescence of extracellular bacterial particles was quenched by adding 0.4% trypan blue to measure only internal fluorescence of ingested bacteria. The acidification of phagosomal compartments was assessed through the uptake of E. coli particles labeled with a low pH–sensitive dye (pHrodo Green; Invitrogen). To control phagocytosis, bacteria were co-labeled with eFluor 670 (eBioscience) in some experiments. Analysis of fluorescence from ingested E. coli was monitored by flow cytometry or fluorescence microscopy. To detect acidic lysosomal compartments, cells were stained with 75 nm LysoTracker red and 1 mM Lysosensor green (all from Invitrogen). Images of live cells containing ingested E. coli were captured at 37°C using a confocal microscope (SP5; Leica Biosystems) with a heated stage.

Westphal A., Cheng W., Yu J., Grassl G., Krautkrämer M., Holst O., Föger N, & Lee K.H. (2017). Lysosomal trafficking regulator Lyst links membrane trafficking to toll-like receptor–mediated inflammatory responses. The Journal of Experimental Medicine, 214(1), 227-244.

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Labeling Apoptotic Endothelial Cells

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Apoptotic mECs prepared as described above were collected and treated with 0.15 mg/ml DNAse I (Sigma-Aldrich) for 15 min in room temperature with gentle shaking. The cells were then incubated with 10μM pHrodo green (Invitrogen, Carlsbad, CA) in 2ml PBS for 30 min with gentle shaking at room temperature. The labeled cells were washed in PBS, centrifuged at 2000 rpm for 5 min, and then resuspended in PBS before being injected into mouse wounds as described below.

Xu M., Chen Z., Chen K., Ma D., Chen L, & DiPietro L.A. (2021). Phagocytosis of apoptotic endothelial cells reprograms macrophages in skin wounds. Journal of immunology and regenerative medicine, 12, 100038.

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Phagocytosis Assays using Flow Cytometry

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We also used two flow-cytometric assays of phagocytosis. First, 50 x 10⁶ AC were labeled with 75μg TAMRA (Invitrogen) in 1 ml PBS and 1 ml LCM for 15 min at 37°C (49 (link)–51 (link)). Labeled AC were washed two times with PBS to remove excess TAMRA. Second, S. aureus pre-labeled with pHrodo-green (Invitrogen) was opsonized by incubation in rabbit anti-staphylococcal mAb (Invitrogen) for 1 h in a 5% CO₂ environment at 37°C, then washed . Mø were plated at 3 x 10⁶ cells/well in 24-well plates. Mø pretreated with cytochalasin D (Sigma) for 1 h before exposure to florescent targets were used to establish baseline florescence. TAMRA-labeled AC or pHrodo-green labeled S. aureus targets were added to Mø for 1 h, then wells were washed briskly with cold PBS to remove unengulfed targets. Mø were detached from culture dishes using the dissociation enzyme TrypLE (Invitrogen), then centrifuged in flow tubes. Internalized fluorescent particles were detected by flow cytometry.

McCubbrey A.L., Nelson J.D., Stolberg V.R., Blakely P.K., McCloskey L., Janssen W.J., Freeman C.M, & Curtis J.L. (2015). miR-34a negatively regulates efferocytosis by tissue macrophages in part via SIRT1. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1366-1375.

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Lysosomal pH Measurement with Dextran Dyes

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Lysosomal luminal acidity was estimated with the fluorescence ratio between a pH-sensitive dye pHrodo™ Green conjugated dextran-10 kd (P35368, Invitrogen) and a pH-insensitive dye CF555 conjugated dextran-10 kd (80112, Biotium). In brief, cells seeded on glass coverslips were loaded with 20 μg/mL of each dextran overnight and chased in medium without dye for 3 hours before imaging. The HEPES buffered DMEM medium without phenol red (21063029, Gibco) was used as the imaging solution to eliminate the short time starvation side effects during imaging process. Cells were washed with the imaging solution and imaged using an inverted microscope (IX81 Olympus) equipped with a 60× objective lens and a temperature-controlled stage. The fluorescence emission excited at 488 nm and 561 nm wavelengths were acquired with an EM-CCD camera (Andor, iXon ultra 897U) controlled by MetaMorph software. The fluorescence intensity of pHrodo™ Green and CF555 were quantified by Fiji/ImageJ.

Hu M., Li P., Wang C., Feng X., Geng Q., Chen W., Marthi M., Zhang W., Gao C., Reid W., Swanson J., Du W., Hume R.I, & Xu H. (2022). Parkinson’s Disease-risk Protein TMEM175 is a Proton-activated Proton Channel in Lysosomes. Cell, 185(13), 2292-2308.e20.

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Quantifying Macrophage ROS Response to Antibiotic Exposure

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Intracellular ROS accumulation in macrophages was detected with the Reactive Oxygen Species Assay Kit (Beyotime) or MitoSOX (Invitrogen) according to the reagent instructions. An overnight culture of S. aureus D36 was labeled with pHrodo-green (Invitrogen) and resuspended in DMEM supplemented with 1% FBS. For live cell imaging, RAW264.7 cells and PMs were incubated with 8 μg mL^-¹ enrofloxacin, followed by S. aureus infection (MOI = 5) for 1 h. After that, cells were washed with prewarmed PBS three times. Cell nuclei, HClO and mtROS were further stained with Hoechst 33342 (Beyotime), APF (Invitrogen) and MitoSOX, respectively, followed by the observation under a Leica SP8 confocal microscope. For relative quantification of ROS, RAW264.7 cells and PMs were seeded at 1 × 10⁶ cells per well in 12-well culture plates or 1 × 10⁵ cells per well in 96-well culture plates. Cells were then treated with ciprofloxacin or enrofloxacin at different concentrations accompanied with 1,000 U mL^-¹ catalase for 12 h. ROS were then labeled with DCFH-DA, and the mean or median fluorescence intensity of FITC was quantified by FCM or an Infinite M200 Microplate reader (Tecan).

Wu Y., Gong X., Shen J, & Zhu K. (2023). Postantibiotic leukocyte enhancement-mediated reduction of intracellular bacteria by macrophages. Journal of Advanced Research, 58, 117-128.

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pH-Dependent Fluorescence Imaging of HeLa Cells

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For fluorescence spectroscopy experiments, HeLa cells (cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum) were seeded at a density of 50.000 cells per well on 24-well plates (Nunclon, Thermo Fisher Scientific) and incubated at 37ºC in a humidified atmosphere with 5% CO₂. Following 48 h incubation period, the cells were washed with PBS (pH 7.4) and incubated at 37°C for 30 min in pHrodo™ Green (Invitrogen, Thermo Fisher Scientific) staining solution (10 μl of pHrodo^TM Green AM added to 100 μl PowerLoad™ concentrate and finally diluted into 10 ml of PBS at pH 7.4). Next, the cells were washed twice and resuspended in PBS adjusted at pH 6.5, 7.4 or 8.0. After a 10 min incubation at room temperature, cells were washed twice again with PBS adjusted at the corresponding pH and fixed with 4% paraformaldehyde adjusted at pH 6.5, 7.4 or 8.0 for 10 min at room temperature (non-fixed cells were incubated in PBS adjusted at the corresponding pH). Then, the cells were washed twice again and incubated for an additional 10 min in PBS adjusted at pH 6.5, 7.4 or 8.0. Immediately afterwards, fluorescence was measured using a BioTek Synergy H1 microplate reader (Agilent Technologies) equipped with Gen5 software (bottom optics positioning, excitation: 500 nm, emission: 535 nm). All samples were prepared in triplicate.

Mir B., Serrano-Chacón I., Medina P., Macaluso V., Terrazas M., Gandioso A., Garavís M., Orozco M., Escaja N, & González C. (2024). Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells. Nucleic Acids Research, 52(6), 3375-3389.

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Antioxidant and Anti-Inflammatory Assays

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Conalbumin (CA), AAPH, and acrolein were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ELISA kits of histamine and IL-4 was acquired from R&D systems (Minneapolis, MN, USA). pHrodo Green was purchased from Invitrogen (Breda, The Netherlands). All other reagents were of analytical grade unless stated otherwise.

Lv L., Ye L., Lin X., Li L., Chen J., Yue W, & Wu X. (2022). Functional and Allergenic Properties Assessment of Conalbumin (Ovotransferrin) after Oxidation. Foods, 11(15), 2308.

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Mast Cell and Dendritic Cell Responses to TM

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The rat mast cell line RBL-2H3 was obtained from the American Type Culture Collection (Bethesda, MD, USA). The cells were cultured as described previously. 29 The release of β-hexosaminidase and histamine from RBL-2H3 cells was measured as a model of IgE-mediated mast cell allergic reaction, 29 using the sensitization of mouse sera and TM or cross-linked TM (500 ng/mL) for 6 h and 15 min at 37 ℃, respectively. The cell supernatant was measured the release levels of β-hexosaminidase and histamine using an ELISA kit (IBL, Hamburg, Germany).
Bone marrow cells from mice were cultured at 10 6 cells/mL in complete RPMI 1640 (Hyclone, Logan, USA) with 10 ng/mL of granulocyte-macrophage colony stimulating factor and 5 ng/mL of interleukin (IL)-4. At day 6, approximately 90% of cells expressed medium-high levels of CD11c and major histocompatibility complex (MHC) class II. To assess protein endocytosis, pHrodo Green (Invitrogen, Life Technologies, Breda, Netherlands)-labeled TM or cross-linked TM was incubated with 10 6 cells for 0, 15, and 30 min. The pHrodo Green-positive DCs were measured by using an automatic microplate reader.

Liu G., Hu M., Sun L.C., Han X., Liu Q., Alcocer M., Fei D., Cao M.J, & Liu G.M. (2017). Allergenicity and Oral Tolerance of Enzymatic Cross-Linked Tropomyosin Evaluated Using Cell and Mouse Models. Journal of agricultural and food chemistry, 65(10).

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Phagocytosis Assay for Peritoneal Macrophages

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Peritoneal macrophages were obtained by intraperitoneal injection of 3% Brewer’s thioglycolate (6 (link)) (Sigma-Aldrich Corp, Darmstadt, Germany) into mice for 72 h. Peritoneal lavage fluid was collected with 5 ml of precooled PBS. Primary peritoneal macrophages were washed twice after lysis of red blood cells, resuspended in medium, and then plated in 6-well plates in DMEM with 10% FBS. We discarded non-adherent cells after 4 h. They were pre-treated with AG1 (3 μM, MedChemExpress, Monmouth Junction, NJ, USA) or PBS (control) for 24 h. Apoptotic cells were labeled with pHrodo™ Green (Thermo Fisher Scientific, Carlsbad, CA, USA), a pH-sensitive phagocytosis-dependent indicator, for 10 min at 37°C, then pHrodo-labeled apoptotic cells were added at a ratio of 1:5 (macrophages:apoptosis cells) at 37°C in DMEM with 10% FBS. After 60 min, macrophages were centrifuged at 1,000 rpm for 5 min, washed twice, resuspended in PBS, and stained with APC anti-mouse F4/80 Antibody (BM8, BioLegend, San Diego, USA); they were later analyzed by flow cytometry.

He D., Mao Q., Jia J., Wang Z., Liu Y., Liu T., Luo B, & Zhang Z. (2022). Pentose Phosphate Pathway Regulates Tolerogenic Apoptotic Cell Clearance and Immune Tolerance. Frontiers in Immunology, 12, 797091.

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Myelin Labeling with CFSE and pHrodo-Green

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Myelin (100 μl) from the above isolation protocol was resuspended in 200 μl of 50 μM CFSE or pHrodo-Green (Thermo Fisher Scientific), incubated at room temperature (RT) for 30 min in the dark, and centrifuged at 14,800 × g for 10 min at 4°C. The pellet containing labeled myelin was then washed three times in 100 mM glycine in PBS and resuspended in sterile PBS to a final concentration of 100 mg/ml. For pHrodo-Green labeling, 18.5 μl myelin was mixed with 25 μl pHrodo-Green and resuspended in 206.5 μl PBS (pH 8). The mixture was incubated in a RT rocker in the dark for 45 min. Myelin mixture was then centrifuged at 4,000 g for 10 min, supernatant was discarded, and resuspended in PBS (pH 7.2).

Gharibani P., Abramson E., Shanmukha S., Smith M.D., Godfrey W.H., Lee J.J., Hu J., Baydyuk M., Dorion M.F., Deng X., Guo Y., Hwang S., Huang J.K., Calabresi P.A., Kornberg M.D, & Kim P.M. (2023). PKC modulator bryostatin-1 therapeutically targets CNS innate immunity to attenuate neuroinflammation and promote remyelination. bioRxiv.

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