Fluorescence activated sorting of antibody-labeled islets using an EPICS Elite ESP flow cytometer

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1999
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Glenn, Justin Garth
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Heath, Carole A.
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A Beckman Coulter EPICS Elite ESP Flow Cytometer with a 400 [Mu]m tip was used to attempt to separate pancreatic tissue from acinar cells following digestion of the pancreas with collagenase. The islets were fluorescently labeled by using A2B5 as a primary antibody, and either a FITC or phycoerythran conjugated antibody as a secondary antibody. A streptavidin/biotin system was also used to fluorescently label the islets. The sorting technique for the EPICS Elite was based on forward-scattering versus side-scattering scheme from which, islets were identified using a forward-scattering versus fluorescence scheme. The attempted sorts were unsuccessful due to excessive fragmentation of the islets. Several tests were performed on the EPICS Elite in order to identify the source of the fragmentation. The tip assembly was identified as this source and two possible causes were postulated. The first suggests that the 400 [Mu]m inner channel is causing the fragmentation due to extended periods of high shear. The second suggest that the hydrodynamic focusing of the sheath fluid when it meets the pancreas solution is great enough to fragment the islets. It was also determined that phycoerythran is better suited to fluorescently label islets for fluorescence-activated sorting due than FITC. The FITC signal is masked by the autofluoreseence of the pancreatic tissue, and therefore not easily recognizable. Also, a Percoll separation was necessary in this process to reduce the number of acinar cells present.
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