Computational protocol: A human endothelial cell-based recycling assay for screening of FcRn targeted molecules

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Protocol publication

[…] HMEC1-hFcRn-EGFP cells were seeded in eight-well NuncTM Lab-TekTM chambered Coverglass imaging dishes (ThermoFisher Scientific), and grown to 50–70% confluency. WT and IHH hIgG1 variants were conjugated with Alexa-647 following the manufacturer’s procedure (Life Technologies). Cells were washed three times with HBSS pH 6.0 and Alexa-647 conjugated antibodies, diluted in HBSS pH 6.0 to the final concentration of 400 nM, were added to the cells and incubated for 4 h before pictures were taken. The cells were then washed three times with HBSS pH 7.4, and incubated in HBSS pH 7.4 for 4 h before pictures were taken. Lysotracker DND-99 (Life Technologies) was added to the cells 30 min before pictures were taken. Confocal images were acquired on an Olympus FluoView 1000 inverted microscope equipped with a PlanApo 60/1.42 oil objective (Olympus). Constant temperature was set to 37 °C and CO2 to 5% by an incubator enclosing the microscope stage. Image acquisition was done by sequential line scanning to eliminate bleed-through. Images were processed and prepared with ImageJ (NIH), Adobe Photoshop and Illustrator (Adobe system Inc). Co-localization was quantified using Imaris spot co-localization software (Bitplane) where the endosome size was set to 1 µm. Data from two independent experiments with 15–20 cells analysed were used for co-localization analysis. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus