The following reagents were purchased from the manufacturers as noted: cholesterol (C8503), sulfatide, PA, ethanolamine, ATP and n-octyl-β-D-glucopyranoside (Sigma); and PI (Echelon); di16:0-PIPs (CellSignals); PS, PE, and PC (porcine brain) for liposome cosedimentation assay, 1,2-dioleoyl-PC, 1,2-dioleoyl-PE, 1,2-dioleoyl-PS, 1,2-dioleoyl-PI, 1,2-dioleoyl-PG, 1,2-dioleoyl-PA, brain polar lipids (porcine), sphingomyelin (porcine brain), cholesterol (ovine wool), C6-NBD-PS and C6-NBD-PE (Avanti Polar Lipids); dithionite (Fisher); CHAPS (Anatrace); and Alexa 594-Tfn, Alexa 594-CTxB, Alexa 555-EGF (Invitrogen). To prepare Alexa 488-Tfn, human holo-Tfn (Sigma) was conjugated with Alexa 488 using Alexa Flour succinimidyl ester (Invitrogen) and then purified by PD-10 desalting columns (GE Healthcare).
Alexa555 egf
Manufactured by Thermo Fisher Scientific
Alexa555-EGF is a fluorescently labeled epidermal growth factor (EGF) product. It is used as a tool for studying EGF receptor (EGFR) expression and dynamics in cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Fm4 64, by Thermo Fisher Scientific (6 mentions)
Dextrin sepharose high performance, by GE Healthcare (2 mentions)
Ni nta agarose, by Thermo Fisher Scientific (2 mentions)
Peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions)
D1817, by Thermo Fisher Scientific (2 mentions)
Alexa 647 egf, by Thermo Fisher Scientific (2 mentions)
Tritc dextran, by Thermo Fisher Scientific (2 mentions)
L7528, by Thermo Fisher Scientific (2 mentions)
E35351, by Thermo Fisher Scientific (2 mentions)
E35350, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 tfp ester, by Thermo Fisher Scientific (2 mentions)
Mbptrap hd 5ml columns, by GE Healthcare (2 mentions)
L leucyl l leucine methyl ester llome, by Merck Group (2 mentions)
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (2 mentions)
Lysotracker, by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Dspe peg biotin, by Avanti Polar Lipids (2 mentions)
Lissamine rhodamine b sulfonyl 18 1 rhod pe, by Avanti Polar Lipids (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Dithionite, by Thermo Fisher Scientific (1 mentions)
5 protocols using alexa555 egf
1
Characterization of Lipid Interactions
2
Quantitative Analysis of EGF Internalization
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Assays were performed essentially as described [31 (link)]. Briefly, transfected HeLa cells were plated onto coverslips the day after transfection, and serum-starved overnight. The following day, medium was replaced with fresh serum-free medium containing 100 ng/mL Alexa555-EGF (Invitrogen) at 4 °C to allow for ligand binding in the absence of internalization. In parallel experiments, control cells were washed with PBS, followed by acid stripping (0.5 M NaCl, 0.2 M acetic acid, pH 2.5, 3 min, 4 °C) to confirm that fluorescent EGF was merely surface-bound under those conditions. After binding of fluorescent EGF at 4 °C, cells were washed twice in ice-cold PBS, and then transferred to prewarmed serum-free medium to allow for uptake, and cells were fixed at the indicated times (10 min and 30 min) to quantify internalized fluorescent EGF. Fixation was performed with 4% PFA in PBS for 15 min at room temperature, cells were softly permeabilized with 0.5% Triton-X100 in PBS for 3 min, and mounted with DAPI. A minimum of 20 and up to 80 independent cells were analyzed for each condition and experiment, and quantification of the number of Alexa555-EGF structures per cell was performed by an observer blind to the conditions [31 (link)].
3
Quantifying EGFR Endocytosis in HeLa Cells
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Assays were performed essentially as described (Rivero-Ríos et al., 2019) .
Briefly, transfected HeLa cells were plated onto coverslips the day after transfection, and serum-starved overnight. The following day, medium was replaced with fresh serum-free medium containing 100 ng/ml Alexa555-EGF (Invitrogen) at 4 ºC to allow for ligand binding in the absence of internalization. In parallel experiments, control cells were washed with PBS, followed by acid stripping (0.5 M NaCl, 0.2 M acetic acid, pH 2.5, 3 min, 4 ºC) to confirm that fluorescent EGF was merely surface-bound under those conditions. After binding of fluorescent EGF at 4 ºC, cells were washed twice in ice-cold PBS, and then transferred to prewarmed serum-free medium to allow for uptake, and cells were fixed at the indicated times (10 min and 30 min) to quantify internalized fluorescent EGF. Fixation was performed with 4% PFA in PBS for 15 min at room temperature, cells were softly permeabilized with 0.5% Triton-X100 in PBS for 3 min, and mounted with DAPI. Minimally 20 and up to 80 independent cells were analyzed for each condition and experiment, and quantification of the number of Alexa555-EGF structures per cell performed by an observer blind to conditions (Rivero-Ríos et al., 2019) .
Briefly, transfected HeLa cells were plated onto coverslips the day after transfection, and serum-starved overnight. The following day, medium was replaced with fresh serum-free medium containing 100 ng/ml Alexa555-EGF (Invitrogen) at 4 ºC to allow for ligand binding in the absence of internalization. In parallel experiments, control cells were washed with PBS, followed by acid stripping (0.5 M NaCl, 0.2 M acetic acid, pH 2.5, 3 min, 4 ºC) to confirm that fluorescent EGF was merely surface-bound under those conditions. After binding of fluorescent EGF at 4 ºC, cells were washed twice in ice-cold PBS, and then transferred to prewarmed serum-free medium to allow for uptake, and cells were fixed at the indicated times (10 min and 30 min) to quantify internalized fluorescent EGF. Fixation was performed with 4% PFA in PBS for 15 min at room temperature, cells were softly permeabilized with 0.5% Triton-X100 in PBS for 3 min, and mounted with DAPI. Minimally 20 and up to 80 independent cells were analyzed for each condition and experiment, and quantification of the number of Alexa555-EGF structures per cell performed by an observer blind to conditions (Rivero-Ríos et al., 2019) .
4
Fluorescent Probes for Cell Imaging
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We obtained FM4-64, LysoTracker, Alexa 647-EGF, Alexa-555 EGF and TRITC-dextran (T13320, L7528, E35351, E35350 and D1817, respectively) from Thermo Fisher Scientific (Waltham, MA), as well as EZ-Link Sulfo-NHS-SS-Biotin, Alexa Fluor 488 TFP ester, Ni-NTA Agarose, 7 kDa MWCO Zeba Spin Desalting Columns and Hoechst 33342. Peroxidase-conjugated secondary antibodies were from Bio-Rad Laboratories (Hercules, CA), MBPTrap HD 5mL columns, Glutathione Sepharose 4B and Dextrin Sepharose High Performance from GE Healthcare (Anaheim, CA), and the cOmplete Protease Inhibitor Cocktail from Roche (Basel, Switzerland). We obtained DOPC, DSPE-PEG-Biotin, DOPE, DOPS and lissamine rhodamine B sulfonyl (18:1) Rhod PE from Avanti Polar Lipids (Alabaster, AL, and the lysosomotropic reagent L-leucyl-L-leucine methyl ester (LLOMe) from Sigma-Aldrich (St. Louis, MO). Other reagents and chemicals were obtained from Sigma-Aldrich (St. Louis, MO)
5
Fluorescent Probes for Cell Imaging
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