lucifer-yellow and Necrosis

The novel morphological characteristics of N9 microglial cells and primary cultured rat microglial cells were examined using the bitotin-IB4 and streptavidin-FITC system. Numerous fine, long processes of both microglial cell preparations formed a network, beginning after 30 min in culture. Dye coupling studies did not show communication between neighbouring cells via the processes in normal conditions. The network of microglial cell processes was well formed into a 'resting state' by 16-24 hr after re-plating. After being challenged by 3 mM ATP the microglial cells were activated, became amoeboid-like cells within 2 hr and finally floated in the culture medium. The complicated network of processes did not retract to the microglial cell body. Flow cytometry analysis showed that the majority of these floating cells were alive and could recover to the resting state after ATP was removed from the culture medium.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cell Line; Cell Size; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Histocytochemistry; Isoquinolines; Microglia; Necrosis; Photomicrography; Rats; Receptors, Purinergic P2; Stimulation, Chemical; Suramin

The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the 'zipper'-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3'-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.

Topics: Androstadienes; Animals; Apoptosis; Cell Line; Cell Line, Tumor; Endocytosis; Fluorescent Dyes; Horseradish Peroxidase; Humans; Isoquinolines; Macrophages; Mice; Microscopy, Electron, Scanning; Necrosis; Phagocytosis; Phosphoinositide-3 Kinase Inhibitors; Pinocytosis; Wortmannin

This paper examines and compares necrosis in human ovarian tissue after conventional slow freezing or vitrification and ensuing xenotranplantation. Slow cryoconserved or vitrified ovarian tissue samples and fresh controls from nine patients were subcutaneously transplanted into SCID mice. The tissue samples were explanted after 6 weeks and the necrotic areas were examined by staining with Lucifer yellow SV. The size of the necrotic areas in parallel cultivated ovarian tissue samples was compared, as was necrosis in cultivated prostate tumour spheroids where the emergence of necrosis and its pathophysiological correlation have been described. Examinations showed no significant rise in the proportion of necrotic areas after slow cryoconservation/transplantation and in the controls (transplanted fresh tissue, not transplanted fresh tissue, long-term culture). The proportion of necrotic areas in the tumour spheroids was significantly higher than in the ovarian tissue. Vitrification could, after these results, be presented as an alternative to conventional slow cryoconservation.

Topics: Adult; Animals; Cell Line, Tumor; Cryopreservation; Female; Fluorescent Dyes; Freezing; Humans; Isoquinolines; Male; Mice; Mice, SCID; Necrosis; Organ Transplantation; Organic Chemicals; Ovary; Prostatic Neoplasms; Time Factors; Transplantation, Heterologous