Osmotic tolerance limits (OTL) of five mouse embryonic stem cell (mESC) lines as determined by plasma membrane integrity. Previously published data for the C57BL/6 mESC line [35] are included for comparative purposes but were not included in statistical analyses. Equal quantities of mESCs were exposed to solutions of 38, 75, 150, 600, 1200, 2400, and 4800 mOsm for 10 minutes at room temperature, and compared with controls exposed to isosmotic solution (285 mOsm) in the same manner. Plasma membrane integrity was assessed by flow cytometry analysis of propidium iodide exclusion. A. Inverted U-shaped osmotic tolerance curves of BALB/c, C57BL/6 (B6), CBA, FVB, and 129R1 (R1) mESC lines. B. OTL, defined as the range of osmolalities in which 80% of cells maintained plasma membrane integrity, for each of 5 different mESC lines (n=3). “0” represents isosmotic (285 mOsm). Distance from isosmotic is in terms of mOsm. Inset numbers indicate the extrapolated mean osmolalities (through linear regression) ± SEM at which 80% of mESCs retained membrane integrity. SEM for all lower OTL was ± 5 mOsm. Different lower case superscripts indicate statistically significant differences in lower OTL (p<0.05).