American Type Tissue Collection

Primary cultures of venous and arterial endothelial cells can be prepared from fresh tissue by well-established methods. However, this is a time consuming and laborious process requiring a good source of tissue. Alternatively, good quality and well-characterized endothelial cells are now readily available from commercial sources, such as the American Type Culture Collection or Clonetics Inc. (Walkersville, MD). The latter currently can supply a range of ECs from different tissues, and from a variety of species. 

STRAINS Available from commercial and private slocks. The American Type Culture Collection has several strains. Although few spawn companies sell strains of L. nuda, tissue and spore cultures are easily obtained from wild specimens. Nevertheless, there are a limited number of productive strains currently in circulation. 

The above listed strains are of European origin. Many strains ofthis species are available from culture banks, including those maintained by the American Type Culture Collection and Pennsylvania State Buckhout Laboratory. Strains are easy to obtain from the spores and tissue of wild specimens. 

The identification of a tissue containing the receptor of interest or the transient or stable cloning and expression in cells is the first step in developing a reliable RRA. While the brain-spinal cord axis represents a major source of receptors, assays based on receptors specific to tissues including gut, platelets, lymphocytes, skeletal muscle, and endocrine organs are well known. Transformed cells are also useful as the point of departure to gather receptors these may be obtained from public sources such as the American Type Culture Collection (Rockville, MD, USA). Cloned and expressed receptors may be obtained on an individual basis as needed. 

The majority of the cell-based assays use established cell lines. The cell line of choice would naturally express receptors of interest that respond to the therapeutic protein product, and can be obtained from commercial sources, American Type Culture Collection (ATCC), for example. Alternatively, if an appropriate commercially sourced cell line cannot be found, one can be engineered to respond to a therapeutic protein (see b.Reporter Gene Assay). Most frequently the cells are stably transfected with a vector containing the desired receptor gene or responsive element of a gene of interest. In rare instances when the cell lines are not available, primary cells separated from blood or tissues (human or animal) can be used to develop bioassays. Primary cells are the last resort because of donor-to-donor variability, accessibility of material, and limited practicality of such assays. 

The isolate of M. roridum used in this study was isolated from diseased tomato tissue provided by B. Bruton, Plant Health and Bioscience, U.S.D.A., ARS, Weslaco,TX. A reference culture has also been deposited at the American Type Culture Collection, Rockville, MD (ATCC 60379). Single spore isolates were maintained on potato-dextrose agar slcints at 24°C under laboratory conditions which resulted in greenish-black masses of conidia 7-10 days after infection. A conidial suspension in sterile distilled water was used to inoculate cultures for trichothecene production. 

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