The VYS cells were rinsed with PBS and resuspended in 300 mL 0.03 g/L trypsin, 10 mM Tris, pH 8.0.
VYS cells ([10.sup.6] cells/mL) were incubated in a Rho 123 solution (100 mg/mL) diluted in DMSO (5 [micro]g/mL) in a 5% C[O.sub.2] incubator for 30 min.
VYS cells were rinsed with PBS and resuspended in 300 [micro]L 0.03 g/L trypsin, 10 mM Tris, pH 8.0.
The pellet of VYS cells was resuspended in PBS at a concentration of 5 x [10.sup.5] cells/mL and incubated for 1 h at 4[degrees]C with 1 [micro]L of specific anti-active caspase-3 antibody with phycoerythrin (PE; Abcam), in the absence or presence of an Ac-Asp-Glu-Val-Asp-OH-specific inhibitor (Abcam).
Comparisons between control and diabetic groups for mean fetal weight and maternal glycemia were performed using Student's t-test and by one-way analysis of variance followed by Tukey's multiple comparison test for significant differences between groups for the characterization of VYS cells, measurement of [DELTA][psi]m, proliferation index, DNA ploidy, cell cycle analysis, and caspase-3 activity.
Expression of the VYS cell markers CD34, VEGFR1, CD115, CD117, CD14, CCR2, CD90, CD44, STRO-1, OCT3/4, and Nanog were detected on VYS cells in both the control and diabetic groups (Figure 1).