droplet of typsin and then plated out at an appropriate density. Again, the polypropylene was checked with a microscope to ensure that all the cells had detached. We found that this protocol did not affect the plating efficiency compared to cells cultured directly in tissue culture flasks. For each survival curve, at least three repeats were irradiated on the same day, with the experiment repeated at least once on a different day. Dosimetry: Linear Energy Transfer calculations, beam homogeneity and beam stability To calculate the dose rate given to cell nuclei, the following formula adapted from Belli et al. (1989) and Combs (2009) was used: et al. (1) where µm-1) is the LET, (particles cm-2 s-1) is the particle flux, and (g cm-3) is L(keV the density of the medium. A slab approximation is used for the cell nucleus even though the cell nucleus can be spherical. A more realistic including spherical cell nuclei would be more representative. However, small variations in the thickness of the nucleus make very little difference to the “volume averaged LET”, as shown below. Hence, such a model makes very little difference to the uncertainties presented in this paper. The volume averaged LET of the particle will depend on the thickness of the nucleus. Concerning V79 cells that are attached to a substrate and lying flat, there is considerable literature on the thickness of the V79 cells nuclei (see Bettega 1998 for experimental work et al. and review ). Here, the value of 6 µm ± 2 µm for flattened nuclei was taken. To our knowledge, for V79 cells in suspension the nuclei dimensions have not been investigated, especially in the current based experimental set-up. Therefore, confocal microscopy (Axiovert 2000, “droplet” Zeiss, Germany) was used to analyse the central thickness of the cells, in 0.5 µm slices, lying on the polypropylene. The cells were fixed with 2% paraformaldehyde, permeabilised with 0.05% triton-X, treated with RNase, stained with To-Pro (Invitrogen, USA) and imaged through the polyproylene on a special jig constructed for the confocal microscope to accommodate custom dishes used in the present experiment. Sampling 100 cells, the average cell nucleus thickness was
droplet of typsin and then plated out at an appropriate density. Again, the polypropylene was checked with a microscope to ensure that all the cells had detached. We found that this protocol did not affect the plating efficiency compared to cells cultured directly in tissue culture flasks. For each survival curve, at least three repeats were irradiated on the same day, with the experiment repeated at least once on a different day. Dosimetry: Linear Energy Transfer calculations, beam homogeneity and beam stability To calculate the dose rate given to cell nuclei, the following formula adapted from Belli et al. (1989) and Combs et al. (2009) was used: (1) where L(keV µm-1) is the LET, (particles cm-2 s-1) is the particle flux, and (g cm-3) is the density of the medium. A slab approximation is used for the cell nucleus even though the cell nucleus can be spherical. A more realistic including spherical cell nuclei would be more representative. However, small variations in the thickness of the nucleus make very little difference to the “volume averaged LET”, as shown below. Hence, such a model makes very little difference to the uncertainties presented in this paper. The volume averaged LET of the particle will depend on the thickness of the nucleus. Concerning V79 cells that are attached to a substrate and lying flat, there is considerable literature on the thickness of the V79 cells nuclei (see Bettega et al. 1998 for experimental work and review ). Here, the value of 6 µm ± 2 µm for flattened nuclei was taken. To our knowledge, for V79 cells in suspension the nuclei dimensions have not been investigated, especially in the current “droplet” based experimental set-up. Therefore, confocal microscopy (Axiovert 2000, Zeiss, Germany) was used to analyse the central thickness of the cells, in 0.5 µm slices, lying on the polypropylene. The cells were fixed with 2% paraformaldehyde, permeabilised with 0.05% triton-X, treated with RNase, stained with To-Pro (Invitrogen, USA) and imaged through the polyproylene on a special jig constructed for the confocal microscope to accommodate custom dishes used in the present experiment. Sampling 100 cells, the average cell nucleus thickness was
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