In Vitro Bioassay of Endotoxin Using Fluorescein as a pH Indicator in a Macrophage Cell Culture System

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH- indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS- detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

Viability of cells in cryopreserved canine cardiovascular organs for transplantation

To determine applicability of the cryopreservation procedure for vessel grafts, the viability of endothelial cells (ECs) among the whole cells in three kinds of organs artery, vein, trachea in mongrel dogs was evaluated on the basis of histological analysis. The Griffonia simplicifolia agglutins-fluorescein isothiocyanate (GSA-FITC) and propidium iodide (PI) double staining methods were combined with flow cytometry (FCM), which was able to simultaneously determine the viability of whole cells and ECs from the same tissue, were performed after harvesting, after antibiotic solution treatment, and after cryopreservation and thawing. In most cases, the viability of ECs is lower than that of whole cells from veins and arteries. The viability of whole cells in veins was maintained until the antibiotic solution treatment and then decreased significantly after cryopreservation and thawing, while the ECs began to decrease significantly after the antibiotic solution treatment and more markedly decreased after thawing. The viability of ECs and whole cells from arteries was similar to that of the veins' conditions. The viability of whole cells from the trachea decreased with a similar pattern to that of the ECs from vessels. In consideration of maintaining cell viability among the three kinds of organs, the viability of arteries was better than that of the others. The cells in the trachea demonstrated a lower viability than the vessels. The effect of antibiotic solution treatment on the reduction of cell viability depends on the treatment time and temperature.