Culturing of Purified ftCells

1. Single rat b-cells do not survive well in suspension. To avoid b-cell losses, FACS-purified rat b-cells are reaggregated in a rotatory shaker for 2 h at 37°C and 5% CO2, in the presence of Ca2+ (see Note 9).

2. Aggregated b-cell clusters can be kept in suspension in serum-free HAM's F10 medium at 37°C and 5% CO2. Depending on the experiment, cultures can be maintained in the absence or presence of 50 iM IBMX. The phosphodiesterase inhibitor mimics paracrine hormone actions on the b-cells, stimulating their in vitro survival and function.

3. b-cells can also be cultured as single cells in poly-D-lysine-coated tissue culture plates with good survival rates. Ninety-six-well plates are coated by incubating the wells with 100 iL of poly-D-lysine (Sigma, 10 ig/mL in water) for 30 min at 30°C, followed by three successive washes with Ham's F10 medium.

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