TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

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.

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Imaging Membrane Curvature inside a FcεRI-Centric Synapse in RBL-2H3 Cells Using TIRF Microscopy with Polarized Excitation

Journal of Imaging ◽

10.3390/jimaging5070063 ◽

2019 ◽

Vol 5 (7) ◽

pp. 63 ◽

Cited By ~ 2

Author(s):

Machado ◽

Bendesky ◽

Brown ◽

Spendier ◽

Hagen

Keyword(s):

Synapse Formation ◽

Immunological Synapse ◽

Immunoglobulin E ◽

Spatial Relationship ◽

Membrane Curvature ◽

Live Cells ◽

Ratio Image ◽

Rat Basophilic Leukemia Cells ◽

High Affinity Ige Receptor ◽

Relationship Of

Total internal reflection fluorescence microscopy with polarized excitation (P-TIRF) can be used to image nanoscale curvature phenomena in live cells. We used P-TIRF to visualize rat basophilic leukemia cells (RBL-2H3 cells) primed with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) coming into contact with a supported lipid bilayer containing mobile, monovalent DNP, modeling an immunological synapse. The spatial relationship of the IgE-bound high affinity IgE receptor (FcεRI) to the ratio image of P-polarized excitation and S-polarized excitation was analyzed. These studies help correlate the dynamics of cell surface molecules with the mechanical properties of the plasma membrane during synapse formation.

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Impaired Intracellular Ca2+Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927605050488 ◽

2005 ◽

Vol 11 (3) ◽

pp. 235-243 ◽

Cited By ~ 4

Author(s):

David M. Plank ◽

Mark A. Sussman

Keyword(s):

Confocal Microscopy ◽

Fluorescence Intensity ◽

Linear Regression Analysis ◽

Data Interpretation ◽

Live Cells ◽

Correction Factors ◽

Pixel Intensity ◽

Scanning Confocal Microscopy ◽

Intracellular Ca ◽

Average Pixel Intensity

Altered intracellular Ca2+dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+concentrations. This study describes methodology to directly measure intracellular Ca2+levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+concentration standards. Titration of Ca2+concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+concentrations. These standard plots can be used to convert transient Ca2+fluorescence obtained with experimental cells to Ca2+concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

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Interactions between semaphorins and plexin/neuropilin receptor complexes in the membranes of live cells

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.100965 ◽

2021 ◽

pp. 100965

Author(s):

Shaun M. Christie ◽

Jing Hao ◽

Erin Tracy ◽

Matthias Buck ◽

Jennifer S. Yu ◽

...

Keyword(s):

Live Cells ◽

Receptor Complexes

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Structural mapping of membrane-bound immunoglobulin E-receptor complexes: use of monoclonal anti-IgE antibodies to probe the conformation of receptor-bound IgE

Biochemistry ◽

10.1021/bi00343a033 ◽

1985 ◽

Vol 24 (22) ◽

pp. 6260-6267 ◽

Cited By ~ 35

Author(s):

David Holowka ◽

Daniel H. Conrad ◽

Barbara Baird

Keyword(s):

Immunoglobulin E ◽

Structural Mapping ◽

Ige Antibodies ◽

Receptor Complexes ◽

Membrane Bound

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A Comparison of Near-Infrared Imaging and Computerized Tomography Scan for Detecting Maxillary Sinusitis

Annals of Otology Rhinology & Laryngology ◽

10.1177/00034894211060623 ◽

2021 ◽

pp. 000348942110606

Author(s):

Mehdi Abouzari ◽

Brooke Sarna ◽

Joon You ◽

Adwight Risbud ◽

Kotaro Tsutsumi ◽

...

Keyword(s):

Maxillary Sinus ◽

Computerized Tomography ◽

Sensitivity And Specificity ◽

Near Infrared ◽

Maxillary Sinusitis ◽

Tertiary Care ◽

Pixel Intensity ◽

Sinus Disease ◽

Nir Imaging ◽

Average Pixel Intensity

Objective: To investigate the use of near-infrared (NIR) imaging as a tool for outpatient clinicians to quickly and accurately assess for maxillary sinusitis and to characterize its accuracy compared to computerized tomography (CT) scan. Methods: In a prospective investigational study, NIR and CT images from 65 patients who presented to a tertiary care rhinology clinic were compared to determine the sensitivity and specificity of NIR as an imaging modality. Results: The sensitivity and specificity of NIR imaging in distinguishing normal versus maxillary sinus disease was found to be 90% and 84%, normal versus mild maxillary sinus disease to be 76% and 91%, and mild versus severe maxillary sinus disease to be 96% and 81%, respectively. The average pixel intensity was also calculated and compared to the modified Lund-Mackay scores from CT scans to assess the ability of NIR imaging to stratify the severity of maxillary sinus disease. Average pixel intensity over a region of interest was significantly different ( P < .001) between normal, mild, and severe disease, as well as when comparing normal versus mild ( P < .001, 95% CI 42.22-105.39), normal versus severe ( P < .001, 95% CI 119.43-174.14), and mild versus severe ( P < .001, 95% CI 41.39-104.56) maxillary sinus disease. Conclusion: Based on this data, NIR shows promise as a tool for identifying patients with potential maxillary sinus disease as well as providing information on severity of disease that may guide administration of appropriate treatments.

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Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1and Gαi-linked G Protein-coupled Receptors

Journal of Biological Chemistry ◽

10.1074/jbc.m112.440057 ◽

2012 ◽

Vol 288 (5) ◽

pp. 3620-3631 ◽

Cited By ~ 24

Author(s):

Christopher P. Vellano ◽

Nicole E. Brown ◽

Joe B. Blumer ◽

John R. Hepler

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Live Cells ◽

Ras Signaling ◽

G Protein Signaling ◽

Protein Signaling ◽

Signaling Complex ◽

And Function ◽

G Protein Coupled

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Bone Morphogenetic Protein Receptor Complexes on the Surface of Live Cells: A New Oligomerization Mode for Serine/Threonine Kinase Receptors

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.1023 ◽

2000 ◽

Vol 11 (3) ◽

pp. 1023-1035 ◽

Cited By ~ 198

Author(s):

Lilach Gilboa ◽

Anja Nohe ◽

Tanja Geissendörfer ◽

Walter Sebald ◽

Yoav I. Henis ◽

...

Keyword(s):

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Live Cells ◽

Type I ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Receptor Complexes ◽

Bmp Receptors ◽

Protein Receptor

The bone morphogenetic proteins (BMPs) play important roles in embryogenesis and normal cell growth. The BMP receptors belong to the family of serine/threonine kinase receptors, whose activation has been investigated intensively for the transforming growth factor-β (TGF-β) receptor subfamily. However, the interactions between the BMP receptors, the composition of the active receptor complex, and the role of the ligand in its formation have not yet been investigated and were usually assumed to follow the same pattern as the TGF-β receptors. Here we demonstrate that the oligomerization pattern of the BMP receptors is different and is more flexible and susceptible to modulation by ligand. Using several complementary approaches, we investigated the formation of homomeric and heteromeric complexes between the two known BMP type I receptors (BR-Ia and BR-Ib) and the BMP type II receptor (BR-II). Coimmunoprecipitation studies detected the formation of heteromeric and homomeric complexes among all the BMP receptor types even in the absence of ligand. These complexes were also detected at the cell surface after BMP-2 binding and cross-linking. Using antibody-mediated immunofluorescence copatching of epitope-tagged receptors, we provide evidence in live cells for preexisting heteromeric (BR-II/BR-Ia and BR-II/BR-Ib) and homomeric (BR-II/BR-II, BR-Ia/ BR-Ia, BR-Ib/ BR-Ib, and also BR-Ia/ BR-Ib) oligomers in the absence of ligand. BMP-2 binding significantly increased hetero- and homo-oligomerization (except for the BR-II homo-oligomer, which binds ligand poorly in the absence of BR-I). In contrast to previous observations on TGF-β receptors, which were found to be fully homodimeric in the absence of ligand, the BMP receptors show a much more flexible oligomerization pattern. This novel feature in the oligomerization mode of the BMP receptors allows higher variety and flexibility in their responses to various ligands as compared with the TGF-β receptors.

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Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.

The Journal of Cell Biology ◽

10.1083/jcb.125.4.795 ◽

1994 ◽

Vol 125 (4) ◽

pp. 795-802 ◽

Cited By ~ 107

Author(s):

J L Thomas ◽

D Holowka ◽

B Baird ◽

W W Webb

Keyword(s):

Cell Surface ◽

Large Scale ◽

Plasma Membranes ◽

Immunoglobulin E ◽

Large Fraction ◽

Surface Antigens ◽

Specific Cell ◽

Cell Surface Antigens ◽

Receptor Complexes ◽

Ige Receptors

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.

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