Lysate preparation from tissues

While the FDA is adopting a cautious approach to cancer vaccine, such as DCVax-Brain, the Swiss Institute of Public Health has conditionally allowed the use of this vaccine by patients. DCVax consists of a patient s dendritic cells that have been pulsed with antigens derived from a tumor cell lysate prepared from surgically resected glioblastoma (brain cancer) tissue. It was developed by a company in the United States but has not yet been approved by the FDA. 

To prepare lysates from (nonstimulated) fibroblasts, cells from one confluent 78-cm2 plate are suspended in 0.15 ml lysis buffer (see below) and lysed by freezing and thawing six times and subsequent centrifugation at 13,000 x for 5 min. An aliquot of 0.05 ml of the supernatant is directly used for the enzyme assay. The preparation of tissue homogenate is described in section 6.1.4.1. GTP cyclohydrolase I, subheading Specimen . 

Tissue lysate (or homogenates), post-mitochondrial supernatants and microsomes offer several practical advantages for the study of xenobiotic metabolism. The principal advantages are that the human tissues provide a complete system containing all the enzymes in ratios found in vivo, and tissue fractions are stable in relatively long-term storage. Within the different types of tissue fractions, microsomes provide an enrichment of the membrane-bound enzymes, and post-mitochondrial supernatants provide a means to study both membrane-bound and soluble enzymes. Tissue fractions are easily prepared from a variety of tissues including human liver and can be cryopreserved for several years. This allows detailed characterization of the tissue prior to use with xenobiotics of unknown routes of metabolism... 

Cell or tissue lysates represent the crudest tissue fraction. Use of lysates is practical when the level of enzyme is relatively high. However, lysates have a tendency to form aggregates, which limits the maximum protein concentration which can be used and also limits the time for which the reaction to be studied is linear. Typically, protein concentrations of less than 3 mg/ml must be used and incubation time is limited to about 30 min. This can be an important limitation for substrates which are metabolized very slowly. In addition, cell fractionation is not limited to primary tissues. Fractions can be prepared from cDNA-expressing cell lines. This can provide a level of convenience to the researcher relative to the demands of maintaining multiple cell lines for extended periods of time. 

Figure 4.7 In-lysate chemical proteomics workflow, (a) Sample preparation from disease-relevant cellular lysate or tissue (b) competition with investigational compound active and inactive of the identical chemotype ...

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

The methods described herein encompass (1) preparation of a whole cell lysate from either cell culture or tissue samples, (2) protein lysate microarray printing, (3) immunostaining, and (4) microarray spot analysis. 

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