ABSTRACT
The development of fluorescent ion-selective indicators for magnesium has provided valuable tools for measuring second-by-second changes in cytosolic magnesium activity. In the course of establishing appropriate protocols for using one of these indicators, mag-fura-2, to measure magnesium activity in BC3H-1 cells and chick ventricular myocytes, many potential pitfalls and limitations of this technique have been encountered and addressed. These observations are presented for the purpose of providing guidelines regarding the appropriate use of this indicator and on factors influencing the interpretation of resulting data.
Subject(s)
Fluorescent Dyes , Fura-2/analogs & derivatives , Heart Ventricles/metabolism , Magnesium/metabolism , Muscle, Smooth/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Chick Embryo , Energy Metabolism , Fura-2/analysis , Heart Ventricles/cytology , Mice , Muscle, Smooth/cytologyABSTRACT
The capillary human tumor clonogenic cell assay (HTCA) has been shown to have important advantages over conventional HTCAs. In the present report, this promising novel HTCA was further optimized and characterized using 46 primary human tumor specimens, 6 human tumor cell lines (1 astrocytoma, 2 colon carcinomas, 1 melanoma), and 2 murine leukemias. Hydrocortisone, epidermal growth factor, heat-inactivated fetal calf serum, and horse serum were investigated for their ability to modulate tumor colony formation in the assay. Critical assay parameters that can affect tumor colony formation, namely, cell seeding density, agarose concentration, culture volume, capillary tube geometry, and capillary tube sealing, were also investigated. The results showed that serum (optimum concentration, 20%) was obligatory for tumor colony formation, and that both epidermal growth factor (50 ng/ml) and hydrocortisone (2.5 ng/ml), although supportive of colony growth, were not absolute requirements. Plating at 2.5-3 x 10(5) cells/ml in a culture volume of 50 microliters/capillary tube and an agarose concentration of 0.2% optimized colony formation (number, size, and distribution of colonies along the capillary tube) by primary human tumor cells. The cell lines generally formed colonies best at lower seeding densities and in lower culture volumes (30 microliters/tube). Colony formation was significantly better in unsealed than in sealed capillary tubes and growth was just as good, and in some cases, better in round capillary tubes than in square ones. Using ovarian carcinoma cells, the Cellscan prototype system was demonstrated as feasible for automated counting and evaluation of tumor colony growth in capillary tubes. A comparison of the capillary HTCA and the agar double-layer assay in Petri dishes produced a median plating efficiency of 0.18 for the capillary HTCA and 0.036 for the Petri dish method. The overall success rate was 77% for the former and 53% for the latter assay.
