Purification of Single Cells and Nonft Cells

1. The dispersed islet cells are washed in isolation medium containing 2.8 mM glucose and submitted to auto-fluorescence-activated cell sorting (FACS) using a FAC-

Fig. 1. FACS analysis of unpurified islets cells examined for their FAD fluorescence and FSC intensity at 2.8 mM glucose. The subpopulation with high FAD and high FSC represents the b-cells, whereas the islet non b-cells are lower in FAD content and cause less FSC.

STAR PLUS. Isolation medium is used as sheath fluid. A 0.22-im filter is put on the sheath tank to remove any particles in the medium.

2. The cells are illuminated with an argon laser (Enterprise II) with 100 mW at 488 nm. The instrument is calibrated according to the manufacturer's guidelines. The fluorescence emission is collected in the FL1 photomultiplier at 510-550 nm (FITC filter, 530-nm bandpass filter). The fluorescence can be taken as a parameter for the cellular FAD content. Rat b-cells have a threefold higher FAD fluorescence than rat non-b-cells at this low glucose concentration. The b-cells are larger than the non-b-cells and have thus a larger forward scatter (FSC). The background signal caused by cell debris is removed by putting a threshold level on FSC. Both FSC and FL1 are linearly amplified.

3. Selection of the appropriate windows allows the simultaneous isolation of single b-cells and single islet non-b-cells. The b-cells are separated on the basis of high FAD fluorescence and high FSC as compared to non-b-cells (Fig. 1). The cells in uncharged droplets are collected as well. They constitute the so-called "middle fraction." The middle fraction is collected in a 50-mL tube, spun down (5 min at 500g), resuspended in low-glucose-containing isolation medium and re-sorted (see Note 8).

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