TIRF Microscopy Image Sequences of FcεRI-Centric Synapse Formation in RBL-2H3 Cells Dataset

Published: 24 June 2020| Version 1 | DOI: 10.17632/6kvzv95w7r.1
Kathrin Spendier


1. Introduction 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. When mast cells contact a monovalent antigen-bearing fluid lipid bilayer, IgE-loaded FcεRI receptors aggregate (forming receptor clusters) at contact points and trigger degranulation and the release of immune activators. After aggregation, micron-sized cluster move by apparently performing diffusion or random walk motion [1] to eventually coalesce to form a big FcεRI-centric receptor patch, called the mast cell synapse [2]. 2. Data Info This data sets contains microscope image sequences of this synapse formation. The data are provided as Image Cytometry Standard (.ics) files with corresponding .ids files. These files can be readily opened with ImageJ (also known as Fiji), see: https://imagej.net/Fiji. Image exposure was 50 ms or 100 ms and pixel size was 167 nm. Images were taken every second. Image exposure is indicated in the folder name. A total of 8 time series or movies of RBL-2H3 cells forming this big FcεRI-centric receptor patch are included in this data set.


Steps to reproduce

For details see reference: K. Spendier, A. Carroll-Portillo, K.A. Lidke, B.S. Wilson, J.A. Timlin, and J.L. Thomas. Distribution and dynamics of rat basophilic leukemia immunoglobulin E receptors (FcεRI) on planar ligand-presenting surfaces. Biophys. J., 99:388–397, 2010. 3. Materials and methods In the experimental model system RBL-2H3 cells were primed with fluorescent anti-dinitrophenyl (anti-DNP) IgE over night and pipette-pressed onto laterally mobile supported lipid bilayers formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti Polar Lipids) with 25 mol% N-dinitrophenylaminocaproyl phosphatidylethanolamine (DNP-Cap PE, Avanti Polar Lipids). Supported lipid bilayers were made by spontaneous liposome fusion [3] as outlined in Ref. [1]. Implementation of a micropipette manipulation technique was necessary to precisely fix the time of first cell contact with a substrate, with an onset precision of ±50 ms [1]. Experimental data was collected with an Olympus IX 71 (Olympus America Inc.) objective-based total internal reflection fluorescence (TIRF) microscope to eliminate background fluorescence from fluorescent receptors away (> 200 nm) from the contact region. A 1.6 × microscope tube lens was used with the 60 × objective to obtain an effective magnification of 96 ×. Images were collected with an EMCCD (iXon 887; Andor Technologies Inc.). The camera was cooled to −100 degrees C (iXon 887) and its gain was set to 100. Sample temperatures were maintained at 37ºC with an objective heater. Images were collected and processed with in-house software implemented in MATLAB (The MathWorks) in conjunction with DIPImage [4] an image processing library. Image exposure was 50 ms or 100 ms and pixel size was 167 nm. Images were taken every second. The microscope point spread function was experimentally determined to be 0.63 micro meters [1]. 4. References [1] Biophys. J., 99:388–397, 2010. [2] J. Immunol., 184:1328–1338, 2010. [3] Langmuir, 25:2986–2993, 2009. [4] DIPImage: a scientific image processing toolbox for MATLAB (http://www.diplib.org/main)


Clustering, Fluorescence, Mast Cell