In order to isolate the cytosolic fractions from northern pike liver, we have cut liver samples to small pieces and then added cooled homogenization buffer [100 mM Tris-HCl/Base (Sigma, pH 7.5 at 4°C) supplemented with reducing agent (1 mM dithiotreitol, Sigma)] (mliver/vbuffer 1:5). The obtained suspensions were homogenized in an ice cooled tube by 10 strokes of Potter-Elvehjem homogenizer (Glas-Col, USA) at 6,000 rpm. The homogenates were then centrifuged at 50,000×g for 2 h at +4°C in the Avanti J-E centrifuge (Beckman Coulter, USA), and supernatants were afterwards stored at -80°C. Thus obtained supernatants (S50) represented soluble tissue fractions, i.e. hepatic cytosols, additionally containing only microsomes (Bonneris et al., 2005) . The entire procedure was performed under ice-cold conditions, and all the necessary preconditions were applied to avoid/limit the equilibrium change among trace elements and biomacromolecules in the cells/tissues (Szpunar et al., 2003; Dragun et al., 2020) .
Potter elvehjem homogenizer
Manufactured by Glas-Col
Sourced in United States
The Potter-Elvehjem homogenizer is a laboratory equipment used for the disruption and homogenization of biological samples, such as tissues or cells. It consists of a glass or Teflon pestle that fits tightly within a glass mortar or tube, allowing for the mechanical disruption of the sample through grinding or shearing forces.
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4 protocols using potter elvehjem homogenizer
1
Isolation of Hepatic Cytosols from Northern Pike
2
Isolation of Hepatic Cytosolic Fractions
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The samples of hepatic tissue were cut into small pieces within glass crystallizing dishes set on ice. Then cooled homogenization buffer 100 mM Tris-HCl/Base (Sigma, pH 8.1 at 4C) supplemented with reducing agent (1 mM dithiotreitol, Sigma) was added into each dish to dilute hepatic tissue (w/v 1:5). Thereafter followed homogenization by application of 10 strokes of Potter-Elvehjem homogenizer (Glas-Col, USA) at 6,000 rpm in an ice cooled tube. The homogenates were then centrifuged at 50,000g for 2 h at 4°C in the Avanti J-E centrifuge (Beckman Coulter, USA). Supernatants (S50) obtained after centrifugation represented water soluble cytosolic tissue fractions containing lysosomes and microsomes (Bonneris et al., 2005) . S50 fractions were separated from centrifuge tubes, transferred to clean tubes and stored in the freezer at -80C.
3
Cytosolic Tissue Fraction Isolation
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The samples of gill tissue were first cut into small pieces. Then cooled homogenization buffer 20 mM Tris-HCl/Base (Sigma, pH 8.6 at 4C) supplemented with reducing agent (2 mM dithiotreitol, Sigma) was added (w/v 1:5). It was followed by homogenization by 10 strokes of Potter-Elvehjem homogenizer (Glas-Col, USA) in ice cooled tube at 6,000 rpm. For better separation, the homogenates were centrifuged subsequently two times in the Avanti J-E centrifuge (Beckman Coulter) at 50,000g for 2 h at 4°C. Supernatant obtained after second centrifugation (S50), which represents water soluble cytosolic tissue fraction containing lysosomes and microsomes (Bonneris et al. 2005) , was separated and stored at -20C for subsequent metal analyses.
4
Cytosolic Fraction Isolation from Trout Liver
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Isolation of soluble cytosolic fraction from S. trutta liver was performed according to the Standard Operational Procedure (1999), which was developed at Norwegian Institute for Water Research in the framework of the Biological Effects Quality Assurance in Monitoring Programmes (BEQUALM) (Dragun et al., 2009) . The samples of hepatic tissue were cut into small pieces. Then cooled homogenization buffer 100 mM Tris-HCl/Base (Sigma, pH 8.1 at 4C) supplemented with reducing agent (1 mM dithiotreitol, Sigma) was added (w/v 1:5), followed by homogenization with 10 strokes of Potter-Elvehjem homogenizer (Glas-Col, USA) in an ice cooled tube at 6,000 rpm. The homogenates were subsequently centrifuged (Avanti J-E centrifuge, Beckman Coulter) at 50,000g for 2 h at 4°C. Soluble cytosolic hepatic fractions, i.e. supernatants obtained after centrifugation of tissue homogenates at 50,000×g, contained cytosolic biomolecules, lysosomes and microsomes, and excluded cell membranes, nuclei, mitochondria and granules (Bonneris et al., 2005; Dragun et al., 2013a; Podrug et al., 2009) .
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