MDCK cells were transfected with mCherry-expressing control or SEPT2 shRNA for 48 h, cultured in low-serum medium (0.5% FBS) for 1 h at 37°C, cooled on ice for 5 min, incubated with 10 µg/ml Transferrin-488 (Thermo Fisher Scientific) in ice-cold low-serum medium for 15 min on ice, washed three times with ice-cold medium, and incubated with medium containing 10% FBS at 37°C for 5, 7.5, and 10 min. Cells were subsequently washed twice with PBS and fixed with 4% PFA.
Transferrin 488
Manufactured by Thermo Fisher Scientific
Transferrin-488 is a fluorescently labeled protein commonly used in cell biology research. It is a conjugate of the iron-binding glycoprotein transferrin and the fluorescent dye Alexa Fluor 488. This product can be utilized to study transferrin receptor-mediated endocytosis and cellular iron uptake.
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Lab products found in correlation
6 protocols using transferrin 488
1
Transferrin Uptake in SEPT2 Knockdown
2
Transferrin Uptake Assay in Cells
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Prior to assay, cells were cultured in serum-free media on coverslips for 24 h. Cells were pulsed with 25 μg/ml transferrin-488 (Thermo Fisher; T13342) in serum-free media on ice for 30 min. Coverslips with cells were transferred to 37°C for 20 min induce transferrin uptake while control coverslips were kept at 4°C. After PBS washes, cells were rinsed for 40 s with acidic stripping solution (500 mM NaCl and 100 mM glacial acetic acid in distilled water) to remove surface-bound transferrin. This was followed by PBS washes and fixing with 3.2% PFA in serum-free media at 4°C for 20 min. After PBS washes, coverslips were mounted in Prolong Gold Antifade reagent with DAPI (Thermo Fisher; P36930), dried overnight, and imaged.
3
HUVEC Cell Uptake Assays
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For BSA and transferrin uptake experiment HUVEC cells were serum starved in EGM-2 medium for 12 hr. For BODIPY uptake cells were serum starved in EGM2 medium containing 1% fatty-acids free BSA (Sigma-Aldrich, A9205) and 10 nM insulin (Sigma-Aldrich, I9278). Cells were incubated for 30 min with serum free medium containing BSA-555 (final concentration 4 µM, Molecular Probes, A34786) or with serum free medium containing 20 mM glucose and 1% BSA for Transferrin-488 (final concentration 50 µg/ml, Invitrogen, T13342) uptake. BODIPY C12–500/510 (final concentration 5 µM, Molecular Probes, D3823) uptake was performed in the above described medium. Stimulation with preclustered Fc proteins was performed simultaneously with the uptake molecule in the specific medium. After 30 minutes cells were washed with the uptake medium and were either fixed with 4% PFA for 10 min, immunostained and imaged, or directly prepared for FACS analysis. Cells were trypsinised (Sigma-Aldrich, T3924) and resuspended in FACS buffer (PBS containing 2% FCS and 2 mM EDTA). Cells were stained with DAPI (Sigma, D9542) for viability. The samples were measured using BD FACSVerse and analysed using FlowJo version 10.3.
4
Quantifying Transferrin Internalization in Fibroblasts
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Confluent fibroblasts in 6‐well plates were washed with PBS, serum‐starved for 2 h followed by feeding with 25 µg/ml Transferrin‐488 (Invitrogen) in DMEM for 30 min at 37°C. The cells were washed briefly with PBS followed by a brief wash with Acidic Wash Buffer (50 mM Glycin 150 mM NaCl pH 3) followed by another wash with Acidic Wash Buffer for 3 min at RT to remove surface‐bound Transferrin. After a final wash with PBS, cells were lysed RIPA buffer (110 μl/well) with protease inhibitors (see below) for 20 min on ice under repeated vortexing. Cells were scraped off and sonicated for 5 min in an ice‐bath. Lysates were freeze‐thawed and subsequently cleared by centrifugation at 20,000 g for 20 min at 4°C. Fluorescence in the supernatant was measured with a fluorometric Microplate Reader Flouroscan FL (Thermo Fisher) excitation filter 485 and emission filter 538. The amount of uptake was normalized to the total protein level (Pierce BCA Protein Assay Kit, Thermo Scientific).
5
Fluorescent Labeling of Transferrin
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Transferrin-488 and transferrin-555 were obtained from Invitrogen and DAPI (4,6-diamidino-2-phenylindol) was used from Carl Roth.
6
Endocytic Trafficking Analysis in COS7 Cells
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