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Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

Cells must be seeded on 0.1% gelatin-coated culture  vessels. To make the coating, dissolve 1.0 g of Gelatin Type A (Sigma G2500) in 1L of distilled water, sterilize by autoclaving. Add 0.4 to 0.5 mL of gelatin solution per cm² of the culture vessel and refrigerate for 10 to 15 minutes. Prior to use remove the gelatin solution and rinse gently with culture medium, adding at least 0.1 to 0.2 ml per cm². Discard the culture medium used for rinsing and use vessels immediately. If required, coated vessels can be prepared in advance and stored at 2 to 8?C no more than 5 days.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh culture medium.  Add appropriate aliquots of cell suspension to new  0.1% gelatin coated culture  vessels (see Handling Procedure for Frozen Cells).
7. Incubate cultures at 37°C.

Kruse F, et al. The cell-specific elastase I enhancer comprises two domains [published erratum appears in Mol. Cell. Biol. 8: 1862, 1988]. Mol. Cell. Biol. 8: 893-902, 1988. PubMed: 3352608

Swift GH, et al. Differential requirements for cell-specific elastase I enhancer domains in transfected cells and transgenic mice. Genes Dev. 3: 687-696, 1989. PubMed: 2744460

Ornitz DM, et al. Elastase I promoter directs expression of human growth hormone and SV40 T antigen genes to pancreatic acinar cells in transgenic mice. Cold Spring Harbor Symp. Quant. Biol. 50: 399-409, 1985. PubMed: 3006998

Rose SD, et al. A single element of the elastase I enhancer is sufficient to direct transcription selectively to the pancreas and gut. Mol. Cell. Biol. 14: 2048-2057, 1994. PubMed: 8114736

Swift GH, et al. An element of the elastase I enhancer is an overlapping bipartite binding site activated by a heteromeric factor. J. Biol. Chem. 269: 12809-12815, 1994. PubMed: 8175694