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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. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A ratio of 1:3 to 1:6 is recommended
Medium Renewal: 2 to 3 times per week

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.

Nucleotide (GenBank) : M92269 Human L-type calcium channel HFCC mRNA, complete cds.

Nucleotide (GenBank) : M92272 Human (clone f21) dihydropyridine-sensitive calcium channel HFCC, 5' end intron 4.

Nucleotide (GenBank) : M92274 Human (clone f39) dihydropyridine-sensitive calcium channel HFCC, 5' end intron 3.

Nucleotide (GenBank) : M92271 Human (clone uf22) dihydropyridine-sensitive calcium channel HFCC, 3' end intron 5.

Nucleotide (GenBank) : M92275 Human (clone f24) dihydropyridine-sensitive calcium channel HFCC, partial intron 1.

Nucleotide (GenBank) : M92270 Human (clone uf51-2) dihydropyridine-sensitive calcium channel HFCC, 5' end intron 2.

Nucleotide (GenBank) : M92273 Human (clone f114-2 ) dihydropyridine-sensitive calcium channel HFCC, 3' end intron 4.

Nucleotide (GenBank) : NM_000719 Homo sapiens calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C), mRNA.

Nucleotide (GenBank) : Z74996 H.sapiens mRNA for voltage-dependent L-type calcium channel, alpha 1C subunit (splice variant).

Nucleotide (GenBank) : Z34809 H.sapiens (HLCC69) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34810 H.sapiens (HLCC70) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34811 H.sapiens (HLCC71) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34812 H.sapiens (HLCC73) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34813 H.sapiens (HLCC74) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34814 H.sapiens (HLCC76) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34815 H.sapiens (HLCC77) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34816 H.sapiens (HLCC78) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34817 H.sapiens (HLCC86) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34818 H.sapiens (HLCC87) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34819 H.sapiens (HLCC88) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34820 H.sapiens (HLCC89) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34821 H.sapiens (HLCC90) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).

Nucleotide (GenBank) : Z34822 H.sapiens (HLCC85) mRNA for voltage-dependent L-type Ca channel alpha 1 subunit (splice variant).