Endosomal pH was measured by a ratiometric method as described previously (Kondapalli et al., 2015 (link)). Cells were starved in serum-free media for 30 min to remove residual transferrin, and incubated with pH-sensitive FITC-transferrin (Thermo Fisher Scientific) (75 μg/mL) and pH insensitive Alexa Fluor 633-transferrin (Thermo Fisher Scientific) (25 μg/mL) at 37°C for 1 h. Transferrin uptake was stopped by placing cells on ice. Cells were then washed twice with ice-cold PBS pH 7.4 to remove excess transferrin, followed by ice-cold PBS pH 5.0 and pH 7.4 to remove surface bound transferrin. Cells were trypsinized and analyzed by flow cytometry. Cells with both FITC-transferrin and Alexa Fluor 633-transferrin were gated and a ratiometric analysis of ~10,000 cells for each biological replicate was used to determine the endosomal pH. A standard curve with different pH values 4.5, 5.5, 6.5, and 7.5 was constructed using the Intracellular pH Calibration Buffer kit (Thermo Fisher Scientific) in the presence of 10 μM K+/H+ ionophore nigericin and 10 μM K+ ionophore valinomycin to equilibrium the endosomal pH with the calibration buffers.
Fitc transferrin
Manufactured by Thermo Fisher Scientific
Sourced in United States
FITC-transferrin is a fluorescent-labeled protein used in various cell biology and molecular biology applications. It serves as a tool for studying cellular processes, such as protein trafficking and endocytosis, by enabling the visualization and tracking of transferrin uptake within cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 633 transferrin, by Thermo Fisher Scientific (3 mentions)
Intracellular ph calibration buffer kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 transferrin, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Celltracker orange cmra dye, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Syto rnaselect green fluorescent cell stain, by Thermo Fisher Scientific (1 mentions)
Methyl β cyclodextrin mβcd, by Merck Group (1 mentions)
Monodansylcadaverine, by Merck Group (1 mentions)
5 n ethyl n isopropyl amiloride eipa, by Merck Group (1 mentions)
Medium 200 lsgs, by Thermo Fisher Scientific (1 mentions)
Rpmi serum free medium, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Cell Applications (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Thermo Fisher Scientific (1 mentions)
5 protocols using fitc transferrin
1
Ratiometric Measurement of Endosomal pH
2
Ratiometric Measurement of Endosomal pH
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Endosomal pH was measured by a ratiometric method as described previously (Kondapalli et al., 2015 (link)). Cells were starved in serum-free media for 30 min to remove residual transferrin, and incubated with pH-sensitive FITC-transferrin (Thermo Fisher Scientific) (75 μg/mL) and pH insensitive Alexa Fluor 633-transferrin (Thermo Fisher Scientific) (25 μg/mL) at 37°C for 1 h. Transferrin uptake was stopped by placing cells on ice. Cells were then washed twice with ice-cold PBS pH 7.4 to remove excess transferrin, followed by ice-cold PBS pH 5.0 and pH 7.4 to remove surface bound transferrin. Cells were trypsinized and analyzed by flow cytometry. Cells with both FITC-transferrin and Alexa Fluor 633-transferrin were gated and a ratiometric analysis of ~10,000 cells for each biological replicate was used to determine the endosomal pH. A standard curve with different pH values 4.5, 5.5, 6.5, and 7.5 was constructed using the Intracellular pH Calibration Buffer kit (Thermo Fisher Scientific) in the presence of 10 μM K+/H+ ionophore nigericin and 10 μM K+ ionophore valinomycin to equilibrium the endosomal pH with the calibration buffers.
3
Transferrin Uptake Imaging in Cells
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MEFs were incubated with 5 µg/ml of Alexa-Fluor-647-transferrin (Thermo, T23366) in the culture medium for 30 min at 4°C or 37°C. Cells were washed twice with cold PBS, fixed using PBS containing 4% paraformaldehyde, washed in PBS and visualized by confocal microscopy. Mean fluorescent signals in each cell were measured using Image J. Cultured neurons were incubated with 50 µg/ml of FITC-transferrin (Thermo, T2871) in the culture medium for 15 min at 4°C or 37°C. Cells were washed twice with cold PBS, fixed using PBS containing 4% paraformaldehyde and 4% sucrose, washed in PBS and then visualized by confocal microscopy. Mean fluorescent intensity was measured along dendrites (100 µm in length) using Image J.
4
HUVEC Apoptosis and Vesicle Generation
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Human umbilical vein endothelial cells (HUVECs) were purchased from Cell Applications, cultured in Medium 200 + LSGS (Gibco, Waltham, MA, USA) on a gelatin-coated surface, and Passage 4 cells were used in experiments. Endothelial cells were exposed to RPMI serum-free medium (Gibco) or Medium 200 + LSGS (depleted of vesicles) for 4 h to produce apoptotic-cell- or healthy-cell-conditioned medium respectively as described previously [13 (link), 15 (link), 18 (link), 19 (link)]. To generate fluorescence-emitting vesicles, HUVECs were stained using CellTracker Orange-CMRA dye or SYTO RNASelect Green Fluorescent cell stain (Molecular Probes, Waltham, MA, USA) for 15 min before treatment. Cytochalasin D, monodansylcadaverine, methyl-β-cyclodextrin (MβCD), and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were purchased from Sigma (Burlington, MA, USA). FITC-Transferrin was obtained from Thermo Fisher (Waltham, MA, USA), and annexin V was obtained from BioLegend (San Diego, CA, USA).
5
Fluorescent Probes for Subcellular Localization
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Cytochalasin
D (#PHZ1063), Alexa
Fluor 633 transferrin (#T23362), FITC transferrin (#T2871), Alexa
Fluor 594 cholera toxin subunit B (#C22842), and Texas Red dextran
(70,000 g/mol, #D1864) were purchased from Thermo Fisher Scientific
(Waltham, Massachusetts, USA). FITC cholera toxin subunit B (#C1655)
was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), and CF488A
dextran (70,000 g/mol, #80117) was purchased from Biotium (Fremont,
California, USA). FuGENE 6 Transfection Reagent was purchased from
Promega (Madison, Wisconsin, USA), and Lipofectamine 2000, Alexa Fluor
594 donkey anti-rabbit IgG (H+L) (#A21207), and Hoechst (#804612)
were purchased from Invitrogen (Waltham, Massachusetts, USA). Bafilomycin
A1 (#88899-55-2) was purchased from Thermo Fisher Scientific (Waltham,
Massachusetts, USA). Alexa Fluor 594 anti-Nuclear Pore Complex Proteins
IgG (#682202) was purchased from Cedarlane (Burlington, Ontario, Canada).
D (#PHZ1063), Alexa
Fluor 633 transferrin (#T23362), FITC transferrin (#T2871), Alexa
Fluor 594 cholera toxin subunit B (#C22842), and Texas Red dextran
(70,000 g/mol, #D1864) were purchased from Thermo Fisher Scientific
(Waltham, Massachusetts, USA). FITC cholera toxin subunit B (#C1655)
was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), and CF488A
dextran (70,000 g/mol, #80117) was purchased from Biotium (Fremont,
California, USA). FuGENE 6 Transfection Reagent was purchased from
Promega (Madison, Wisconsin, USA), and Lipofectamine 2000, Alexa Fluor
594 donkey anti-rabbit IgG (H+L) (#A21207), and Hoechst (#804612)
were purchased from Invitrogen (Waltham, Massachusetts, USA). Bafilomycin
A1 (#88899-55-2) was purchased from Thermo Fisher Scientific (Waltham,
Massachusetts, USA). Alexa Fluor 594 anti-Nuclear Pore Complex Proteins
IgG (#682202) was purchased from Cedarlane (Burlington, Ontario, Canada).
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