Effects of deuterium oxide on cell growth and vesicle speed in RBL-2H3 cells
- Subject Areas
- Biophysics, Biotechnology, Cell Biology
- RBL-2H3 cells, Flow Cytometry, deuterium oxide, single-particle tracking, TIRF microscopy, microtubule-dependent vesicle transport
- © 2014 Kalkur et al.
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2014. Effects of deuterium oxide on cell growth and vesicle speed in RBL-2H3 cells. PeerJ PrePrints 2:e416v2 https://doi.org/10.7287/peerj.preprints.416v2
For the first time we show the effects of deuterium oxide on the cell growth and vesicle transport in rat basophilic leukemia (RBL-2H3) cells. RBL-2H3 cells cultured with 15 moles/L deuterium showed decreased cell growth which was attributed to cells not doubling their DNA content. Experimental observations also showed an increase in vesicle speed for cells cultured in deuterium oxide. This increase in vesicle speed was not observed in deuterium oxide cultures treated with a microtubule-destabilizing drug suggesting that deuterium oxide affects microtubule-dependent vesicle transport.
This is version 2 of a submission to PeerJPrePrints # 2014:06:2277:0:0:PREPRINT:P, and this version of the manuscript was accepted for publication in PeerJ.
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