Antibody against cytokeratin 3/12 was purchased from Abcam (Cambridge, MA). Anti-acetylated tubulin (0.05 µg/mL), and chemicals including cytochalasin D, cytochalasin B, bafilomycin A1, 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA), and IPA-3 were purchased from Sigma-Aldrich. Antibodies to clathrin and dynamin 2 were obtained from Abcam. Anti-adaptor protein 2 subunit alpha 1 (AP2A1) antibody and Transferrin-Alexa Fluor 568 were obtained from Thermo Fisher Scientific. Antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), EEA1, and lysosome-associated membrane protein 1 (LAMP1) were obtained from Santa Cruz Biotechnology (Dallas, TX). Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA). Lipofectamine RNAiMAX was obtained from Thermo Fisher Scientific.
Transferrin alexa fluor 568
Manufactured by Thermo Fisher Scientific
Sourced in United States
Transferrin Alexa Fluor 568 is a fluorescently labeled protein used for labeling and tracking transferrin in biological samples. It consists of the transferrin protein conjugated to the Alexa Fluor 568 fluorescent dye. The core function of this product is to enable the visualization and detection of transferrin in various experimental and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Dmpe peg2000, by NOF corporation (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
5 n ethyl n isopropyl amiloride eipa, by Merck Group (1 mentions)
Anti acetylated tubulin, by Merck Group (1 mentions)
Ipa 3, by Merck Group (1 mentions)
Cy3 dye, by GE Healthcare (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Lamp1, by Santa Cruz Biotechnology (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Digitonin, by Merck Group (1 mentions)
Insulin, by AstraZeneca (1 mentions)
Pioglitazone, by AstraZeneca (1 mentions)
Egf alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Cholesterol assay kit, by Abcam (1 mentions)
8 protocols using transferrin alexa fluor 568
1
Endocytic Pathways Characterization
2
Gavage-Based Gut Lumen Trafficking
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5-dpf larvae were gavaged as previously described (Cocchiaro and Rawls, 2013 (link)). Larvae were gavaged with 4 nl of a mixture containing 10 µM LysoTracker Red (ThermoFisher Scientific), 500 µM FM4-64 (ThermoFisher Scientific), or 1.25 mg/ml Transferrin-AlexaFluor-568 (ThermoFisher Scientific) in PBS plus 0.05% phenol red. For gavage pulse-chase analysis, larvae were gavaged with one of the solutions above, transferred back to egg water to recover for 90 min, and then gavaged with excess PBS (1–2 pulses of 5–10 nl each), which displaces contents of the intestinal lumen through the cloaca. Flushing of the intestinal lumen was visually assessed by confirming thorough displacement of phenol red from the gut. The chase period proceeded for 90 min, and then larvae were processed for sectioning and confocal imaging. In other experiments, larvae were gavaged with inhibitors, ionophores, or control vehicles dissolved in egg water (pH 7.5) plus 0.05% phenol red. Concentrations and durations used are indicated in the figure legends.
3
Transferrin Uptake Dynamics in Cells
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Transfected cells were rested for 30 min in DMEM medium without serum. Cells were then incubated with 25 μg of transferrin Alexa Fluor 568 (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures. Extracellular Tf was removed as previously described for EGF internalization.
4
Liposome Delivery of Fluorescent-Labeled Biomolecules
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FBS Superior, dexamethasone, 3-isobutyl-1-methyxanthine, insulin, and pioglitazone were provided by Astra Zeneca. P/S, transferrin Alexa Fluor 568 (T23365; Invitrogen), EGF Alexa Fluor 488 (E13345; Invitrogen), LDL-pHrodo Red (L34356; Invitrogen), LDL-Alexa Fluor 488 (homemade) were as previously reported (Franke et al., 2019 (link)). pH calibration buffers were from Invitrogen (P35379). LNPs were prepared using the cationic ionizable lipids MC3, L608, ACU5, ACU22, MOD5, and L319 (AstraZeneca) and the helper lipids cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; CordenPharma), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DMPE-PEG2000; NOF Corporation), and contained CleanCap eGFP mRNA (5-methoxyuridine, cat. no. L-7201) and/or CleanCap Cy5 eGFP mRNA (5-methoxyuridine, batch no. WOTL18871, cat. no. L-7701; TriLink Bio Technologies). Formaldehyde was from Merck and digitonin from Sigma-Aldrich.
5
Cholesterol Trafficking Assay Protocol
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The antibodies used in this study are listed in Appendix Table S2 . Lysotracker (#L7528) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). LDL (#437644) was purchased from EMD Millipore Corporation. Filipin (#F4767), cholesterol–methyl‐beta‐cyclodextrin complex (#C4951‐30MG), 2‐hydroxypropyl‐beta‐cyclodextrin (#332593), U18666A (#U3633), and unlabeled transferrin (#T0665) were obtained from Sigma. Transferrin Alexa Fluor 568 was purchased from Invitrogen. The Cholesterol Assay Kit (#K623‐100) was obtained from BioVision.
6
Fluorescent Probes for Organelle Staining
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Sterolight, whose synthesis and characterization was described previously45 (link), was provided by Michal Jurášek (ICT, Prague). Alexa Fluor 568-transferrin (23365), ER Tracker Blue-White DPX (E12353), MitoTracker Deep Red FM (M22426), LysoTracker Red DND-99 (L7528), Rhodamine B-labelled dextran (RhoDex; 10 kD) (D1824), Hoechst 33324 (62249), Draq5 (62251) were from Thermo Fisher Scientific. Lipi-Red (LD03) from Dojindo Molecular Technologies. Filipin III (F4767) was from Merck.
7
Immunofluorescence and Western Blot Analysis
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Rabbit anti-Vimentin (D21H3, immunofluorescence IF 1:100, western blotting WB 1:100) and rabbit anti-Cortactin (H222, IF 1:200, WB 1:1000) were from Cell Signaling Technology. Mouse anti-Paxillin (5H11, IF 1:200, WB 1:1000) and TopFluor® Cholesterol (810255P) were from Merck Millipore. Mouse anti-KDEL (10C3, IF 1:300), Dyngo® 4a (ab120689) and Hoechst 33258 (ab228550) were from Abcam. Alexa Fluor™ 568 Phalloidin (A12380), Alexa Fluor™ 647 Phalloidin (A22287), Oregon Green™ 488 conjugate (G13186), Fluo4-AM (F-14201), ER-Tracker™ Red (E34250), LysoTracker™ Green DND-26 (L7526), LysoTracker™ Red DND-99 (L7528), Alexa Fluor 568 Transferrin (T23365), DAPI (D3571) and fluorescent secondary antibodies were from ThermoFisher Scientific. Peroxidase-conjugated secondary antibodies were from ThermoFisher Scientific and Sigma. SiR-actin (SC001) was from Spirochrome. GM6001 MMP inhibitor (BML-EI300-0001) was from Enzo Life Sciences. Mouse anti-α-Tubulin (DM1a, IF 1:100, WB 1:1000), methyl-β-cyclodextrin (mβCD; C4555), cholesterol-Water Soluble (C4951), cytochalasin D (cytoD) from Zygosporium mansonii (C8273) and growth factor-reduced ECM gel from Engelbreth-Holm-Swarm mouse sarcoma (E6909) were from Sigma-Aldrich.
8
Visualizing Endocytic Dynamics in Giardia
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Approximately 1 × 105 GFP-EHD1- or mock-GFP-expressing trophozoites in 1 mL BI-S-33 were placed on a 35-mm collagen-coated glass-bottom culture dish (MatTek Corporation, Ashland, MA) for 15 min to allow cell attachment. The medium was removed and replaced with 1 mL of BI-S-33 supplemented with either 2 mg/mL RITC-dextran (molecular weight [MW] = 70 000; Sigma-Aldrich, USA) or 100 μg/mL Alexa Fluor 568-transferrin (Thermo Fisher Scientific, USA). Chase was performed for up to 30 min for live imaging. For IFA, fixation was conducted with HA-EHD1 and mock-HA strains after 0, 30, 60, and 120 min of addition of either RITC-dextran or Alexa Fluor 568-transferrin. Live images were captured using an LSM780 confocal laser scanning microscope (Carl Zeiss Microscopy, Germany), as the cells were being incubated at 35°C using a temperature-controlled stage plate (Carl Zeiss Microscopy, Germany).
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