Tissue preparation protocols washing procedures

The volume of the tissue should not exceed 10% of the volume of TRI REAGENT . Alternatively, rather than using the whole spleen directly, splenocytes can first be prepared from spleen by using standard procedures described for preparation of hybridoma production (see Chapter 1) before total RNA extraction. A spleen from an immunized mouse contains approx. 5 X 10 to 2 X 10 lymphocytes. For total RNA extraction, substitute 10 lymphocytes for a spleen and simply add 10 ml of TRI REAGENT (1 ml of reagent for 10 cells) to the sterile PBS-washed cell pellet. Allow incubation at room temperature for 5 min for lysis, and proceed to Protocol 2. step 2. 

Due to the fact that MT is a native protein, its separation from real samples is closely connected with an isolation process. This procedure is being imderstood as a complex of individual steps, which start with pretreatment methods involving extraction and purification and continue with separation techniques prior the MT detection. At the beginning of the quantification of MT, it is necessary to prepare samples (from cells or tissues) according the following protocol (Figure 3). In the case of the cells that were treated with metal ions, samples should be washed with buffer solution to remove residual culture medium and metals adsorbed on the surface of the cells. 

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