In order to study internalization, 1A3 was conjugated with pHrodo iFL Red dye using pHrodo iFL Red Microscale Protein Labeling Kit (P36014, Thermo Fisher Scientific). MCF7 cells were seeded in 12 mm Nunc Glass Bottom Dish, at a density of 1 × 105 cells/dish. After allowing the cells to attach overnight, the cells were incubated for 60 min with 1 mg/mL Hoechst dye for nuclei staining and LysoTracker® Green DND-26 (Thermo Fisher Scientific) for lysosomal labeling. The cells were then incubated with 1A3-pHrodo (2 μg/mL) for 20 min at 37°C. After washing the wells with PBS, the cells were supplemented with 1× live-cell imaging solution (Molecular probes, Thermo Fisher Scientific). Fluorescence images were acquired at 30 min intervals for 2.5 h with the Olympus 1X71 inverted fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). TAPI-2 (20 μM)—pre-treatment was performed overnight, with DMSO serving as a control.
Phrodo ifl red microscale protein labeling kit
Manufactured by Thermo Fisher Scientific
The PHrodo iFL Red Microscale Protein Labeling Kit is a product designed for labeling proteins with a pH-sensitive fluorescent dye. The kit enables the fluorescent detection of proteins in various biological applications.
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Lab products found in correlation
Lysotracker green dnd 26, by Thermo Fisher Scientific (2 mentions)
Molecular probe, by Thermo Fisher Scientific (1 mentions)
Nunc glass bottom dish, by Thermo Fisher Scientific (1 mentions)
Phrodo red succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Incucyte zoom live cell system, by Sartorius (1 mentions)
Aliphatic amine latex beads, by Thermo Fisher Scientific (1 mentions)
Syn per reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Opera phenix high content screening system, by PerkinElmer (1 mentions)
Harmony analysis software, by PerkinElmer (1 mentions)
Dmem without phenol red, by Thermo Fisher Scientific (1 mentions)
Immu mount, by Thermo Fisher Scientific (1 mentions)
4 protocols using phrodo ifl red microscale protein labeling kit
1
Visualizing 1A3 internalization in MCF7 cells
2
Phagocytosis Assay of Microglia
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Mice were perfused transcardially with phosphate buffer before brain dissection. Then whole brain was homogenized in Syn-PER Reagent (ThermoFisher Scientific) and synaptosomes were pelleted after centrifuge at 15,000g. Purified synaptosomes were quantified and then labeled with pHrodo Red dye by using pHrodo iFL Red Microscale Protein Labeling Kit (ThermoFisher Scientific).
Aliphatic amine latex beads (3 µm, Thermofisher) were incubated with 3 mg/ml mouse IgG (Thermofisher) on a rotator overnight at 4 °C to allow proper binding. After washing to remove any unbound IgG, IgG-opsonized latex beads were labeled with pHrodoRed, succinimidyl ester (Thermofisher) for 1 h at room temperature, followed by repeated wash to remove free dye.
Microglia treated with LPS or poly(I:C) for 24 h were fed with pHrodo-labled synaptosomes, pHrodo-labeled E. coli (Sartorius), or pHrodo-labeled IgG-opsonized latex beads. Fluorescence was monitored every half an hour for 7.5 h by IncuCyte Zoom live-cell system (Sartorius).
Aliphatic amine latex beads (3 µm, Thermofisher) were incubated with 3 mg/ml mouse IgG (Thermofisher) on a rotator overnight at 4 °C to allow proper binding. After washing to remove any unbound IgG, IgG-opsonized latex beads were labeled with pHrodoRed, succinimidyl ester (Thermofisher) for 1 h at room temperature, followed by repeated wash to remove free dye.
Microglia treated with LPS or poly(I:C) for 24 h were fed with pHrodo-labled synaptosomes, pHrodo-labeled E. coli (Sartorius), or pHrodo-labeled IgG-opsonized latex beads. Fluorescence was monitored every half an hour for 7.5 h by IncuCyte Zoom live-cell system (Sartorius).
3
Lysosomal Trafficking of HER3-DXd
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The lysosomal trafficking of 0.1, 1, and 10 nM HER3-DXd into the HER3 transfectants was determined using HER3-DXd labeled with a pH-sensitive dye, pHrodo (pHrodo iFL Red Microscale Protein Labeling Kit #P36014, Thermo Fisher Scientific Inc). The cells were treated with the pHrodo-labelled HER3-DXd, 100 nM LysoTracker Green DND-26, and 100 ng/mL Hoechst 33342 (both Thermo Fisher Scientific Inc). Fluorescence emission images were then collected at 30-minute intervals for up to 12 hours using a 403 confocal Opera Phenix high-content screening system (PerkinElmer Co., Ltd., Waltham, MA). Using Harmony analysis software (PerkinElmer Co., Ltd.), the level of lysosomal trafficking was expressed as the number of dots per cell and the dot signal intensity. Dot signal intensity was calculated by subtracting the average dot intensity value at t = 0 from each signal intensity value. The level of lysosomal trafficking was also expressed as a trafficking index, calculated by multiplying the number of dots per cell by the delta dot signal intensity.
4
Visualization of SHBG Endocytosis in Fibroblasts
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Thirty-five thousand cells of genital skin fibroblasts were seeded on glass coverslips (diameter: 13 mm) in DMEM without phenol red (Thermo Fisher Scientific) + 1% Pen/Strep + 5% FBS. Human recombinant Sex hormone-binding globulin (SHBG; R&D Systems) was labeled with pHrodo™ iFL Red Microscale Protein Labeling Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.
The day after seeding the cells were incubated with DMEM without phenol red + 1% Pen/Strep and 5% FBS for 24 h at 37°C and 5% CO 2 .
The cells were then serum-starved overnight and incubated with ECGreen-Endocytosis Detection fluorescent dye (Dojindo, Europe) diluted 1:4000 in medium. ECGreen-labeled fibroblasts were incubated with pHrodo-labeled SHBG for 10 min. Afterwards cells were washed once with phosphate-buffered saline and fixed in 4% paraformaldehyde for 5 min. After additional three washing steps, the cells were stained with DAPI, washed again and mounted on a slide with Immu-Mount (Thermo Fisher Scientific).
The day after seeding the cells were incubated with DMEM without phenol red + 1% Pen/Strep and 5% FBS for 24 h at 37°C and 5% CO 2 .
The cells were then serum-starved overnight and incubated with ECGreen-Endocytosis Detection fluorescent dye (Dojindo, Europe) diluted 1:4000 in medium. ECGreen-labeled fibroblasts were incubated with pHrodo-labeled SHBG for 10 min. Afterwards cells were washed once with phosphate-buffered saline and fixed in 4% paraformaldehyde for 5 min. After additional three washing steps, the cells were stained with DAPI, washed again and mounted on a slide with Immu-Mount (Thermo Fisher Scientific).
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