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This cell line is useful for studying glucagon biosynthesis and alpha cell sensitivity to cytokines.

alphaTC1 clone 9 is a pancreatic alpha cell line cloned from the alpha TC1 cell line which was derived from an adenoma created in transgenic mice expressing the SV40 large T antigen oncogene under the control of the rat preproglucagon promoter.

This cell line is heterozygous for MHC alleles derived from both parental strains used in construction of the transgenic mice C57BL/6J(H-2b and DBA/2J(H-2d).

H-2b

H-2d

glucagon

glucagon

Though the parental alphaTC1 cell line produces glucagon and considerable quantities of insulin and preproinsulin mRNA, the clonal line is more differentiated and produces glucagon but not insulin or preproinsulin mRNA.

Rat recombinant gamma-interferon or mouse recombinant interleukin-1 individually inhibited glucagon synthesis in alphaTC1 clone 9 cells but did not cause inhibition of DNA synthesis.

The combination of these cytokines caused marked inhibition of DNA and glucagon synthesis in alphaTC1 clone 9 cells.

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
NOTE: Warm all solutions to 37°C prior to use.

1. Transfer all medium and floating cells from flask to a 50 mL centrifuge tube.
2. Adherent cells are removed using Cell Dissociation Buffer (an enzyme free buffer; Invitrogen, Catalog No. 13150-016). Add 5 mL of diluted cell dissociation buffer per 75 cm² flask and gently rock flask to bathe the cells at room temperature for 1 to 2 minutes.
3. Allow the flask to remain at room temperature for 1 to 5 additional minutes until cells have detached from the flask.
4. Firmly tap the flask against palm of hand to dislodge cells.
5. Add 10 mL of fresh medium per 75 cm² flask and triturate up and down directing the stream along the growth surface of the flask to dislodge the cells and break up some of the clumps.
6. Transfer these cells to the centrifuge tube from Step 1. Centrifuge at 125 x g for 5 to 10 minutes. Remove medium and resuspend pellet in fresh complete medium.
7. Add appropriate aliquots of cell suspension to new culture vessels.
8. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended.

Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase

Temperature: 37°C

Atmosphere: air, 90%; carbon dioxide (CO₂), 10%