Scanning X-ray microanalysis of microcarrier cultured endothelial cells: elemental changes during the transition to confluency and the effect of ionophore A23187.
Porcine endothelial cells were grown on microcarrier beads and examined by scanning electron microscopy (SEM) at various times after initiation of culture. Total cell coverage on the bead surface varied from mean values of approximately 7% (3h) to 80% (96h). Beam penetration into the subcellular matrix presents a major problem with SEM X-ray microanalysis of microcarrier cultured cells and necessitates the use of an accelerating voltage not exceeding 10kV. At this voltage and below, X-ray contribution from elements present in the microcarrier bead has minimal effect on the determination of cell elemental levels. Washing the cells with 0.15M sucrose was the least perturbing of the rinsing techniques investigated, removing surface culture medium but not internal diffusible ions. X-ray microanalysis revealed detectable levels of Na, P, S, Cl, K and Ca in the cells, with well-marked changes from initial attachment to confluency. The level of K decreased from approximately 1.0% at 3h to 0.4% at 24h, with a corresponding decrease in the K/Na ratio. This unexpectedly low level of K was invariably observed after 24h, and is a genuine feature of established microcarrier culture. The effect of ionophore A23187 was determined at the 3h culture stage, and resulted in significant increases in the concentration of divalent cations (Mg2+, Ca2+), monovalent ions (Na+, Cl-) and a decrease in the level of K+.