TIRF Microscopy Image Sequences of Fluorescent IgE-FcεRI inside a FcεRI-Centric Synapse in RBL-2H3 Cells Dataset

Published: 22 June 2020| Version 3 | DOI: 10.17632/ckj5p6tjyk.3
Contributors:
Kathrin Spendier,
Rachel Drawbond

Description

Total internal reflection fluorescence (TIRF) microscopy was used to image the IgE-FcεRI receptor signaling complex in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer, modeling an immunological model synapse. Fluorescent immunoglobulin E (IgE488) concentrations ranged from 10% to 100%, in 10% increments and IgEdark that decreased from 90% to 0% by increments of 10%. To label, the IgE488 and IgEdark were added to the RBL-2H3 cells in suspension the day prior to imaging and allowed to incubate overnight. Immediately before experimental data was taken, cells were removed from the suspension dish and resuspended in Hank’s buffer. Cells of Sample 1 were kept in Hank’s buffer for a few minutes before cells were added to the SLB. Cells from Sample 2 were kept in Hank’s buffer for about half an hour before cells were added to the SLB. Cells from Sample 3 were kept in Hank’s buffer for about one hour before cells were added to the SLB. Multiple image sequenceswere taken for each of these ten conditions with camera exposure of 5 ms per image and pixel size of 107nm. The fluorescent labeling efficiency of IgE488 was 1.02 ± 0.08 mole fluorescent dye per mole protein. The dataset contains corresponding dark images and TIRF illumination profile images. The camera gain g was 0.006 photo electrons/ADU and the readout noise Nread was 0.529 photo electrons. The data are provided as OME-TIFF (.ome.tif), a life sciences file format. Information about the OME-TIFF (.ome.tif) file format information as well as imaging software supporting it can be found here: https://docs.openmicroscopy.org/ome-model/5.6.3/ome-tiff/

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Steps to reproduce

see paper "TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells" by Rachel Drawbond and Kathrin Spendier: Drawbond, R.; Spendier, K. TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells. Data 2019, 4, 111.

Institutions

University of Colorado at Colorado Springs

Categories

Receptor, Fluorescence Imaging, Mast Cell

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