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Supplemental Information

MOVIE S1

Supplemental movie S1 shows diI-stained, IgE-loaded RBL-2H3 cells that settled onto glass under gravity. Cells were unstimulated or stimulated with multivalent DNP25- BSA for more than 26 min and imaged with TIRF microscopy at 20 frames/s. Time t = 0 min is approximately 5 min after initial cell-substrate contact. Top row: cell before DNP25-BSA stimulation (left panel), after approximately 20 min DNP25-BSA stimulation (middle panel), and after approximately 26 min DNP25-BSA stimulation (right panel). Bottom row: resting (unstimulated) cell in contact with the glass substrate at t = 0 min (left panel), at t = 20 min (middle panel), and at t = 26 min (right panel).

DOI: 10.7287/peerj.preprints.416v2/supp-1

Figure S1

To confirm incorporation of diI into the cell plasma membrane and membrane-bound organelles, diI-stained RBL-2H3 cells were imaged using a confocal microscope. Cell samples were maintained at 37oC using an objective heater. For confocal imaging, a Leica TCS SP5 confocal laser scanning microscope with a 63× oil immersion objective was used. diI-labeled cells were excited with 543 nm laser light in confocal microscopy. Appropriate filter settings were used to collect fluorescence. Figure S1 depicts a confocal z-stack montage of a diI-labeled RBL-2H3 cell and shows that diI labels cell membrane barriers, including the cell plasma membrane and membrane-bound organelles such as the nucleus, lysosomes, and endosomes. This figure shows a confocal z-stack montage of a diI-labeled RBL-2H3. All panels correspond to individual confocal z-slices starting with the z-slice of the cell in contact with the glass substrate, panel A, and going up to the top of the cell, panel X. The z-step size was set to 692 nm. Scale bar represents 10 µm.

DOI: 10.7287/peerj.preprints.416v2/supp-2

Cell count and viability data for figure 1

Data in form of XLSX files

file name description:

Alive and Dead Cell Count: data for figure 1A

Cell Viability: data for figure 1B

DOI: 10.7287/peerj.preprints.416v2/supp-3

Flow Cytometry raw data for figure 2

.LMD files are the Flow Cytometry raw data for figure 2. .PDF files are overview pages showing analyzed data with the applied gatesFile name description:day_one_H2O: one-day old culture with 0 mol/L deuterium oxide day_four_H2O: four-day old culture with 0 mol/L deuterium oxide day_one_D2O: one-day old culture with 15 mol/L deuterium oxide day_four_H2O: four-day old culture with 15 mol/L deuterium oxide

DOI: 10.7287/peerj.preprints.416v2/supp-4

Additional Information

Competing Interests

The authors declare that there were no competing financial, professional or personal interests that might have influenced the performance or presentation of the work described in this manuscript.

Author Contributions

Roshni S Kalkur conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper, these authors contributed equally to this work..

Andrew C Ballast conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper, these authors contributed equally to this work..

Ashley R Triplett conceived and designed the experiments, performed the experiments, analyzed the data, reviewed drafts of the paper.

Kathrin Spendier conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

University of Colorado at Colorado Springs Institutional Review Board, IBC 13-001

Data Deposition

The following information was supplied regarding the deposition of related data:

TIRF data uploaded to FigShare:

http://dx.doi.org/10.6084/m9.figshare.1059555

http://dx.doi.org/10.6084/m9.figshare.1060175

http://dx.doi.org/10.6084/m9.figshare.1060377

http://dx.doi.org/10.6084/m9.figshare.1060390

http://dx.doi.org/10.6084/m9.figshare.1060416

http://dx.doi.org/10.6084/m9.figshare.1060428

http://dx.doi.org/10.6084/m9.figshare.1060436

Funding

This work was supported by the University of Colorado, Colorado Springs BioFrontiers Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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