Message-Id: <199801230107.MAA25808@loki.mbcmr.unimelb.edu.au> X-Sender: paukovic@mail X-Mailer: Windows Eudora Light Version 1.5.2 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: cyto-inbox From: Geza Paukovics <paukovic@mbcmr.unimelb.edu.au> Subject: 3 questions Status: RO Geza Paukovics <paukovic@mbcmr.unimelb.edu.au> asked: >Recently I was asked if it possible to sort intracellular particles, eg. >mitochondria etc.. Has anyone done this..how good are the yields? is there a >protocol which may be employed or are there any helpful references...I have >a dual laser FACStar Plus. There are so many questions....non-specific >staining? Do I have to change the nozzle size (currently 75um), do I have to >alter the drop drive frequency and to what optimal frequency? are there >alternate methods other than flow? will I be battling with high abort rates? It's probably possible to sort some organelles. Nuclei are a pretty easy bet. If you count a chromosome as an organelle, these too are sortable. Ira Mellman's group has also succeeded in isolating endosomal organelles using an electrophoresis technique. I heard him speak in 1996, about the time that Immunity 1996 Mar;4(3):229-239 came out, so you might want to start with that reference. If you can do it by electrophoresis, you can probably also do it by flow. Now, mitochondria may be another story. The information that I will give you here is second-hand, from a friend who worked in David Clayton's group. It's my understanding that mitochondria have never been isolated from cellular lysates. Furthermore, some confocal microscopy studies were performed with anti-mitochondrial stains, and discrete dots were not ob- served in the cytoplasm. Rather, there were broad tubules and intercon- necting blobs. This has led some people to suggest that the mitochondrial ultrastructure is not a series of discrete organelles after all, but rather a reticulated network of membranes. The pictures of tidy, oval-shaped mitochondria that we have all seen are derived from electron micrographs, which are two-dimensional pictures of the cell. The researchers who favor the hypothesis that the mitochondrial ultrastructure is a network say that the electron microscope pictures only emphasize those parts of the network that are sliced cross-wise, and that the parts that are fractured in a length-wise manner are missed.
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