Document Type

Article

Publication Date

1-1-2006

Department

Biology

Abstract

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors (Steinberg et al., 1987; Sung et al., 1985), and with sustained application of ATP it has been shown that dyes as large as 831 daltons can permeate the cell membrane (Steinberg et al, 1987). The current study explores the kinetics of loading α,α-trehalose (342 daltons) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor (Steinberg et al., 1987). Cells that were incubated at 37 ̊C in a 50 mM phosphate buffer (pH 7.0) contailing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of ~50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 ̊C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 oC (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 oC to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49% ± 12 % by Trypan Blue exclusion, 56 ± 20% by Alamar BlueTM assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.

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