Transferrin uptake was measured by using pH-insensitive Alexa Fluor 488-transferrin as described previously (Kondapalli et al., 2015 (link)). Cells were starved in serum-free media for 30 min, followed by incubation with Alexa Fluor 488-transferrin (Thermo Fisher Scientific) (100 μg/mL) for 30 min at 37°C. For uptake kinetics, cells were incubated with Alexa Fluor 488-transferrin for 2, 5, 10, 15, and 30 min. For transferrin recycling kinetics, cells were incubated with Alexa Fluor 488-transferrin for 30 min, washed twice with ice-cold PBS pH 7.4 and incubated in normal media for 0, 5, 10, 15, and 30 min. Cells were then washed 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. Transferrin uptake or recycling were quantified by analyzing ~10,000 cells using flow cytometry. Surface accessible transferrin receptors was determined by cell surface biotinylation. Starved cells were incubated with biotin conjugated transferrin (50 μg/mL, Thermo Fisher Scientific) for 30 min at 4°C and washed twice with ice-cold PBS pH 7.4 to remove excess transferrin. Cells were then lysed for western blot.
Alexa fluor 488 transferrin
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
Alexa Fluor 488-transferrin is a fluorescently labeled protein compound. It is used as a tool in scientific research to visualize and track the movement and localization of transferrin, a protein involved in iron transport, within cells.
Automatically generated - may contain errors
Lab products found in correlation
Biotin conjugated transferrin, by Thermo Fisher Scientific (2 mentions)
Fitc dextran, by Thermo Fisher Scientific (1 mentions)
Fortessa flow cytometer, by FlowJo (1 mentions)
Facscanto 2, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dobutamine, by Merck Group (1 mentions)
Ml141, by Bio-Techne (1 mentions)
Sp2 confocal microscope, by Leica (1 mentions)
Heparin sodium salt, by Merck Group (1 mentions)
Wortmannin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cosmo Bio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Methyl β cyclodextrin mβcd, by Merck Group (1 mentions)
Eht1864, by Cayman Chemical (1 mentions)
1 1 1 3 3 3 hexafluoro 2 propanol hfip, by Fujifilm (1 mentions)
Secinh3, by Cayman Chemical (1 mentions)
G lisa, by Cytoskeleton (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Phalloidin alexa fluor phalloidin 647, by Thermo Fisher Scientific (1 mentions)
12 protocols using alexa fluor 488 transferrin
1
Quantifying Transferrin Uptake and Recycling
2
Quantifying Cargo Internalization Kinetics
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For experiments utilizing pHrodo-labeled cargo, cells were washed and incubated with 125 μL cargo-containing 1:1 PBS/DMEM media for 15 minutes at room temperature. pHrodo Green Dextran MW10000 (ThermoFisher, Cat No P35368) and pHrodo Red Transferrin (ThermoFisher, Cat No P35376) were used at 30 μg/mL. After incubation, media were removed, cells washed, and 200 μL of 1:1 DMEM/PBS was added. Cells were then incubated at 37°C for the specified time (10, 20, 30, or 45 minutes). Cells were washed and 300 μL of PBS with 2% FBS and 0.02% sodium azide was added. Cells were removed via cell scraper and fluorescence data was collected using an LSRII or Fortessa flow cytometer and analyzed using FlowJo 9.8.8. For experiments utilizing FITC-Dextran (ThermoFisher, Cat#D1820), AlexaFluor488-Transferrin (ThermoFisher, Cat No T13342) or AlexaFluor488 labeled IAV PR8, cells were washed and incubated with 125 μL of cargo-containing 1:1 PBS/DMEM media. Cells were placed at 37°C for the specified time (5, 10, 15, 30 or 45 minutes) without preincubation at room temperature. Cells were harvested at time points using a cell scraper, and placed on ice as described above. Flow cytometry data were collected using an LSRII or Fortessa flow cytometer and analyzed using FlowJo 9.8.8.
3
Quantifying Transferrin Uptake and Recycling
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Transferrin uptake was measured by using pH-insensitive Alexa Fluor 488-transferrin as described previously (Kondapalli et al., 2015 (link)). Cells were starved in serum-free media for 30 min, followed by incubation with Alexa Fluor 488-transferrin (Thermo Fisher Scientific) (100 μg/mL) for 30 min at 37°C. For uptake kinetics, cells were incubated with Alexa Fluor 488-transferrin for 2, 5, 10, 15, and 30 min. For transferrin recycling kinetics, cells were incubated with Alexa Fluor 488-transferrin for 30 min, washed twice with ice-cold PBS pH 7.4 and incubated in normal media for 0, 5, 10, 15, and 30 min. Cells were then washed 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. Transferrin uptake or recycling were quantified by analyzing ~10,000 cells using flow cytometry. Surface accessible transferrin receptors was determined by cell surface biotinylation. Starved cells were incubated with biotin conjugated transferrin (50 μg/mL, Thermo Fisher Scientific) for 30 min at 4°C and washed twice with ice-cold PBS pH 7.4 to remove excess transferrin. Cells were then lysed for western blot.
4
Transferrin Uptake in HepG2-NTCP-C4 Cells
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HepG2-NTCP-C4 cells were cultured with 100 μM silibinin for 2 h, and cultured with 5 μg/ml Alexa Fluor 488 transferrin (Thermo Fisher Scientific) with 2% albumin for 15 min. They were fixed and observed as described above. Similarly, after adding transferrin, the cells were collected after treatment with 0.25% trypsin (Thermo Fisher Scientific), and quantification of fluorescence-labeled transferrin in the cells was performed by flow cytometry using FACS Canto II (BD Biosciences, Franklin Lakes, NJ).
5
Visualizing C. burnetii trafficking
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Vero cells seeded on glass coverslips at 4 x 104 cells per well in 24-well plates were infected at an MOI of 100. Two dpi cells were treated with DMSO or CK-666 for 24 hr. Cells were incubated with 25 μg/ml of Alexa Fluor-488 transferrin (ThermoFisher, T13342) for 1 hr, washed three times with PBS, fixed with 4% PFA, then fluorescently immunostained for CD63 and C. burnetii.
6
Effects of Cdc42 Inhibition on Cell Signaling
7
Transferrin Internalization Assay
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Two healthy control cell lines (C1 and C2) and one patient‐derived cell line expressing the p.R465W mutation were used 48 h after siRNA transfection. Cells were cultured in DMEM without FCS for 45 min at 37°C. AlexaFluor488‐Transferrin (Life Technologies, France) was added at 40 μg/ml, and cells were incubated at 37°C for 15 min. Cells were washed three times with PBS and fixed in paraformaldehyde 4% at room temperature for 15 min. Stacks of cell images (0.5 μm interval) were obtained using a Leica SP2 confocal microscope. Fluorescent‐positive surface was quantified on stack projection using ImageJ software and normalized to the total cell surface.
8
Probing Amyloid-beta Internalization Mechanisms
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FITC-labeled Aβ42 was purchased from GL Biochem Ltd. (Shanghai, China); TMR-labeled Aβ42, FITC-dextran (MW: 70,000), a marker for the macropinocytosis pathway, FITC-Cholera Toxin B subunit (CTB), a marker of Lipid raft, heparin sodium salt an inhibitor of HSPGs, and methyl-β-cyclodextrin (MβCD) that can disrupt lipid raft, were obtained from Sigma-Aldrich (St. Louis, MO, USA); ethylisopropyl amiloride (EIPA) a blockef of macropinocytosis, SecinH3, an indirect inhibitor of Arf6, Grassofermata (NAV 2729) a direct inhibitor of Arf6, and EHT1864, a Rac1 inhibitor of were purchased from Cayman chemical (Ann Arbor, MI, USA); dynasore (ab120192), a dynamin inhibitor was from Abcam (Cambridge, MA, USA), Alexa Fluor 488 transferrin and phalloidin were obtained from Invitrogen (Carlsbad, CA, USA); wortmannin, a phosphoinositide 3-kinase inhibitor, Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium, phenol red-free Ham’s F12 Medium, 0.25% trypsin-EDTA, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan); fetal bovine serum was from COSMO BIO (Tokyo, Japan); and Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI), were from Dojindo (Kumamoto, Japan). G-LISA (ELISA-based GTPase activation assay) kits obtained from Cytoskeleton (Denver, CO, USA) were used to measure the activities of Rac1 and Arf6.
9
Integrin-Transferrin Dynamics in Microchannel
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Before injection in the microchannels, clusters are incubated in DMSO for control condition or with 10 μM cRGD for 30 min. Transferrin (Alexa Fluor 488-transferrin; 5 μg/ml; Invitrogen, T13342) is also added for all conditions to reveal the endosomal fraction. Clusters are then injected in PEG-coated microchannels in full media containing Transferrin and cRGD or DMSO and incubated overnight. Integrin αV and transferrin are then imaged using a Spinning Disk CSU-W1 microscope (Yokogawa) with a Prime 95B sCMOC camera.
10
Embryonic Actin Cytoskeleton Visualization
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Embryos were removed from decidua and extra-embryonic membranes discarded. Transferrin alexa Fluor 488 (ThermoFisher) was prepared at 25 μg/ml in DMEM. Embryos were incubated at 37 °C for 5 min. After washing, embryos were fixed in 4% paraformaldehyde overnight. Actin staining was performed by incubating embryos in Phalloidin (Alexa Fluor Phalloidin 647, ThermoFisher) overnight. Images were collected by confocal microscopy (Zeiss LSM 710) and processed using ImageJ software.
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