Abstract
Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE488) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.
Highlights
Understanding cellular transmembrane signaling is critical to almost all aspects of cell biology
Our principal interest is immune signaling by mast cells
DNP-bovine serum albumin (BSA) concentration-dependent degranulation of cells that were suspended in Hanks buffer for an hour prior to performing the degranulation assay decreased by 38%–65% compared to cells that were kept in cell media until the degranulation assay was performed
Summary
Understanding cellular transmembrane signaling is critical to almost all aspects of cell biology. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per percent IgE488. DNP-bovine serum albumin (BSA) concentration-dependent degranulation of cells that were suspended in Hanks buffer for an hour prior to performing the degranulation assay decreased by 38%–65% compared to cells that were kept in cell media until the degranulation assay was performed. This decrease in degranulation may be an indication that RBL-2H3 cells become stressed when suspended in Hanks buffer for an extended time. The dataset can be used to investigate the concentration fluctuation of the IgE-FcεRI receptor signaling complex within the FcεRI-centric synapse to determining the receptor signaling complex size [30]
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