Clathrin and dynamin were inhibited using Pitstop 2 ™ (Ascent Scientific) and Dynasore (Merck), respectively. Cells were pretreated with either 30 μM of Pitstop 2 ™ or with 150 μM of Dynasore for 30 min at 37 °C, followed by 10 min of incubation with MOA-Cy3 and Transferrin-Alexa 488 (Life Technologies). Cells were then fixed in 4% PFA at 4 °C for 10 min and imaged on a confocal microscope.
Transferrin alexa 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Transferrin-Alexa 488 is a fluorescently labeled protein used in various research applications. It is composed of the transferrin protein conjugated with the Alexa Fluor 488 dye. This product can be used to study protein localization, trafficking, and interactions in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Dynasore, by Merck Group (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Deltavision omx sr system, by GE Healthcare (1 mentions)
Leica tcs sp5 confocal laser scanning microscope, by Leica Microsystems (1 mentions)
Lsm 780 laser scanning confocal microscope, by Zeiss (1 mentions)
Alexa 568 transferrin, by Thermo Fisher Scientific (1 mentions)
Alexa488 dextran, by Thermo Fisher Scientific (1 mentions)
5 protocols using transferrin alexa 488
1
Visualizing Clathrin and Dynamin Inhibition
2
Fluorescent Labeling of Cellular Uptake
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Cells were incubated with 10 μg ml−1 of Transferrin-Alexa-488, 5 μg ml–1 of Cholera Toxin subunit B-Alexa-488 (Life Technologies), or 500 μg ml−1 of Dextran-FITC (Sigma-Aldrich) during 30 min at 37 °C in SF medium. Cells were detached, washed in PBS and resuspended in PBS containing 2% formaldehyde (Sigma) and 0.02% sodium azide (Merck). Cell-associated fluorescence was acquired on an Accuri C6 Flow cytometer and analysed using Accuri C6 software (BD Biosciences).
3
Visualizing OROV Viral Factory Formation
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Cells were seeded on 13-mm-diameter coverslips and fixed at the indicated times p.i. and processed for Immunofluorescence assay as previously described [54 (link)]. For dsRNA detection, using J2 antibody, samples were processed according to [55 (link)]. To detect recycling endosomes, we incubated serum starved OROV-infected HeLa cells with transferrin-Alexa 488 (life technologies) at 20 μg/mL in Opti-MEM for 30 min at 4°C. Then, cells were washed with ice-cold PBS to remove non-attached transferrin and incubated for an additional 1 h at 37°C. Cells were analyzed on a Zeiss confocal laser scanning microscope (LSM) 780 (Zeiss, Jena, Germany) or a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). Post-acquisition image processing and colocalization analysis were accomplished as previously described [52 (link)]. To determine the area of Vfs we used the analyze particles tools of the Fiji software [56 (link)], setting the lower and upper threshold levels of each image to 30,000 and 63,000, respectively. Alternatively, fixed cells images were acquired by a DeltaVision OMX SR system (GE Healthcare Life Sciences, Issaquah, WA, USA) by structured illumination microscopy (SIM). After deconvolution, projection of z-stacks (0,125 μm interval each), 3D sections or 3D reconstruction images were analyzed by Fiji software [56 (link)].
4
Transferrin Uptake and Recycling Assay
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For the transferrin uptake and recycling assays, WT, Eipr1 KO, and Ccdc186 KO 832/13 cells were seeded onto 24-well plates and grown in complete media until they reached confluence. For the uptake, cells were placed in warm uptake medium (serum-free RPMI + 1% BSA + 25 mM HEPES + 50 µg/ml Alexa 488 transferrin; Invitrogen; #T13342) for the indicated times (Figure 8A , top). For the recycling, cells were placed in warm uptake medium for a 25-min pulse. Medium was then exchanged for complete RPMI medium and cells were chased for the indicated times.
Cells were then transferred to ice, washed twice with ice-cold PBS, and detached on ice with 10 mM EDTA in PBS for 30 min to 1 h with manual agitation and gentle pipetting. Detached single cells were transferred into a microcentrifuge tube and fixed at 4°C with 4% PFA (final concentration ∼3.5%) for 10 min on a Nutator to avoid clumping. Fixed cells were washed with PBS and analyzed by FACS using a LSRII (BD Biosciences). Data were analyzed using FlowJo.
For transferrin immunostaining, cells were seeded onto coverslips in 24-well plates for 24–48 h. Cells were then placed in warm uptake medium for a 25-min pulse (serum-free RPMI + 1% BSA + 25 mM HEPES + 50 µg/ml Alexa 568 transferrin; Invitrogen; #T23365), washed twice with PBS, and fixed with 4% PFA as already described.
Cells were then transferred to ice, washed twice with ice-cold PBS, and detached on ice with 10 mM EDTA in PBS for 30 min to 1 h with manual agitation and gentle pipetting. Detached single cells were transferred into a microcentrifuge tube and fixed at 4°C with 4% PFA (final concentration ∼3.5%) for 10 min on a Nutator to avoid clumping. Fixed cells were washed with PBS and analyzed by FACS using a LSRII (BD Biosciences). Data were analyzed using FlowJo.
For transferrin immunostaining, cells were seeded onto coverslips in 24-well plates for 24–48 h. Cells were then placed in warm uptake medium for a 25-min pulse (serum-free RPMI + 1% BSA + 25 mM HEPES + 50 µg/ml Alexa 568 transferrin; Invitrogen; #T23365), washed twice with PBS, and fixed with 4% PFA as already described.
5
Co-localization of Aptamers and Organelles
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For co-localization experiments cells were co-incubated with 250 nM folded aptamers and 50 μg/ml of Alexa488-transferrin (Invitrogen™ by Life Technologies; Carlsbad, MA, USA) or 200 μg/ml Alexa488-dextran (Thermo Fisher Scientific Inc. Waltham, MA, USA). Subsequent washings, fixations and quenching steps were performed as described above.
For identification of endosome-like structures a co-staining was performed using aptamers or antibodies and Alexa488-transferrin. Cells were co-incubated with primary antibodies or aptamers as described above with 50 μg/ml Alexa488-transferrin for 1 hour at 37°C and 5% CO2 in complete DMEM. Washing, fixation, permeabilisation and incubation of secondary antibodies were performed as described above.
For identification of endosome-like structures a co-staining was performed using aptamers or antibodies and Alexa488-transferrin. Cells were co-incubated with primary antibodies or aptamers as described above with 50 μg/ml Alexa488-transferrin for 1 hour at 37°C and 5% CO2 in complete DMEM. Washing, fixation, permeabilisation and incubation of secondary antibodies were performed as described above.
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