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TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3
Rachel Drawbond1,2, Kathrin Spendier2,3
1UCCS Center of the Biofrontiers Institute, University of Colorado at Colorado Springs, Colorado Springs, CO 80918, USA.
Data
|July 25, 2020
Summary
This study visualizes the IgE-RC signaling complex in RBL-2H3 cells using TIRF microscopy. Increased fluorescent IgE concentrations directly correlated with higher average pixel intensity in synapses.
Area of Science:
- Cell biology
- Immunology
- Biophysics
Background:
- Total internal reflection fluorescence (TIRF) microscopy is vital for observing live-cell receptor dynamics.
- The IgE-FcεRI receptor signaling complex (IgE-RC) plays a crucial role in immune responses.
- Supported lipid bilayers and RBL-2H3 cells model immunological synapses for studying cellular interactions.
Purpose of the Study:
- To present a dataset of TIRF microscope image sequences for studying the IgE-RC in RBL-2H3 cells.
- To analyze the relationship between fluorescent IgE concentration and receptor signaling within an artificial immunological synapse.
- To investigate the impact of Hanks buffer incubation time on RBL-2H3 cell degranulation.
Main Methods:
- Utilized TIRF microscopy to image IgE-RCs in RBL-2H3 cells interacting with a supported lipid bilayer.
- Varied fluorescent anti-DNP IgE concentrations (10% to 100%) in cell priming.
- Collected image stacks, dark images, and TIRF illumination profiles for data correction and analysis.
Main Results:
- Average pixel intensity within FcεRI-centric synapses increased linearly with fluorescent IgE concentration (0.42 ± 0.02 photo electrons/pixel/% IgE488).
- Data processing involved background subtraction, illumination correction, and unit conversion.
- Prolonged exposure to Hanks buffer inhibited RBL-2H3 cell degranulation, assessed via β-hexosaminidase activity.
Conclusions:
- The dataset provides valuable resources for quantitative analysis of IgE-RC dynamics in model synapses.
- Fluorescent IgE labeling allows for direct correlation with receptor complex intensity in TIRF imaging.
- Cellular buffer incubation conditions significantly affect RBL-2H3 cell exocytosis, impacting experimental outcomes.
