Islet Purification

Conditions of centrifugal elutriation allow elimination of particles smaller than 100 im in diameter. The technique involves the use of a 10X elutriator rotor installed in a JB6 centrifuge.

1. The pancreatic digest is suspended in the mixing chamber that is connected to a flask containing isolation medium.

2. With the elutriator running at 250 rpm, the cellular material is perfused into the elutriation chamber at a rate of 230 mL/min. Particles larger than 100 im in diameter are retained in the elutriation chamber; smaller fragments leave the rotor and are discarded.

3. After disposal of 800-900 mL eluent, the elutriation chamber is disconnected from the circuit and the centrifugation speed is turned down to zero. While the centrifuge is slowing down, the content of the elutriation chamber is collected (see Note 4).

4. The elutriation is stopped when 500 mL eluent has been collected. The fraction is examined under an inverted dissection microscope. Clean islets are hand-picked with an elongated Pasteur pipet (see Notes 5 and 6).

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