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).
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.
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
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