Ca SKi

Product Information Sheet for ATCC® CRM-CRL-1550™ ATCC® (American Type Culture Collection) 800-638-6597 or 703-365-2700 P.O. Box 1549 Fax: 703-365-2750 Manassas, VA 20108 USA E-mail: tech@atcc.org www.atcc.org Or contact your local distributor. Doc ID: 27624 Revision: 1 Effective Date: 08/31/2009 - 1 OF 3 - Cell Line Designation: Ca SKi ATCC® Catalog No. CRM-CRL-1550™ Table of Contents: • Cell Line Description • Biosafety Level • Use Restrictions • Handling Procedure for Frozen Cells • Handling Procedure for Flask Cultures • Medium Renewal • Complete Growth Medium • Cryoprotectant Medium • References • Warranty • Specific Batch Information Cell Line Description Organism: Homo sapiens (human) Tissue: cervix; from metastatic site: small intestine; epidermoid carcinoma Age: 40 years Gender: female Ethnicity: Caucasian Morphology: epithelial Growth Properties: adherent Isoenzymes: G6PD, B Products: beta subunit of human chorionic gonadotropin (hCG); tumor associated antigen DNA Profile: (STR analysis) Amelogenin: X CSF1PO: 10 D13S317: 8,12 D16S539: 11,12 D5S818: 13 D7S820: 8,11 TH01: 7 TPOX: 8 vWA: 17 Depositor: R.A. Pattillo Comments: This cell line was established from cells from a metastasis in the small bowel mesentery. The cells are reported to contain an integrated human papillomavirus type 16 genome (HPV-16, about 600 copies per cell) as well as sequences related to HPV-18. Biosafety Level: 2 Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 4th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 1999. The entire text is available online at www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm. Use Restrictions These cells are distributed for research purposes only. ATCC recommends that individuals contemplating commercial use of any cell line first contact the originating investigator to negotiate an agreement. Third party distribution of this cell line is discouraged, since this practice has resulted in the unintentional spreading of cell lines contaminated with inappropriate animal cells or microbes. Handling Procedure for Frozen Cells To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –70°C. Storage at –70°C will result in loss of viability. SAFETY PRECAUTION: ATCC highly recommends that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris. 1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes). 2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions. Product Information Sheet for ATCC® CRM-CRL-1550™ ATCC® (American Type Culture Collection) 800-638-6597 or 703-365-2700 P.O. Box 1549 Fax: 703-365-2750 Manassas, VA 20108 USA E-mail: tech@atcc.org www.atcc.org Or contact your local distributor. Doc ID: 27624 Revision: 1 Effective Date: 08/31/2009 - 2 OF 3 - 3. Transfer the vial contents to a centrifuge tube containing 9.0 ml complete culture medium and spin at approximately 125 xg for 5 to 7 minutes. Discard supernatant. 4. Resuspend the cell pellet with the recommended complete medium and dispense into a 25 cm2 culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). 5. Incubate the culture at 37°C in a suitable incubator. A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet. Handling Procedure for Flask Cultures The flask was seeded with cells (see specific batch information) grown and completely filled with medium at ATCC to prevent loss of cells during shipping. 1. Upon receipt visually examine the culture for macroscopic evidence of any microbial contamination. Using an inverted microscope (preferably equipped with phase- contrast optics), carefully check for any evidence of microbial contamination. Also check to determine if the majority of cells are still attached to the bottom of the flask; during shipping the cultures are sometimes handled roughly and many of the cells often detach and become suspended in the culture medium (but are still viable). 2. If the cells are still attached, aseptically remove all but 5 to 10 ml of the shipping medium. The shipping medium can be saved for reuse. Incubate the cells at 37°C in a 5% CO2 in air atmosphere until they are ready to be subcultured. 3. If the cells are not attached, aseptically remove the entire contents of the flask and centrifuge at 125 xg for 5 to 10 minutes. Remove shipping medium and save. Resuspend the pelleted cells in 10 ml of this medium and add to 25 cm2 flask. Incubate at 37°C in a 5% CO2 in air atmosphere until cells are ready to be subcultured. Subculturing Procedure Volumes used in this protocol are for a 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 1. Remove and discard culture medium. 2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which 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. Subcultivation Ratio: 1: 3 to 1: 8 6. Incubate cultures at 37°C. Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005. Medium Renewal Two to three times weekly Complete Growth Medium The base medium for this cell line is ATCC-formulated RPMI- 1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: • fetal bovine serum to a final concentration of 10% This medium is formulated for use with a 5% CO2 in air atmosphere. ATCC tested fetal bovine serum is available as ATCC ® Catalog No. 30-2020 (500mL) and ATCC® Catalog No. 30-2021 (100mL). Cryoprotectant Medium Complete growth medium described above supplemented with 5% (v/v) DMSO. Cell culture tested DMSO is available as ATCC Catalog No. 4- X. Product Information Sheet for ATCC® CRM-CRL-1550™ ATCC® (American Type Culture Collection) 800-638-6597 or 703-365-2700 P.O. Box 1549 Fax: 703-365-2750 Manassas, VA 20108 USA E-mail: tech@atcc.org www.atcc.org Or contact your local distributor. Doc ID: 27624 Revision: 1 Effective Date: 08/31/2009 - 3 OF 3 - Additional Information Additional product and technical information can be obtained from the catalog references and the ATCC Web site at www.atcc.org, or by e-mail at tech@atcc.org. References (additional references may be available in the catalog description at www.atcc.org) Pattillo RA, et al. Tumor antigen and human chorionic gonadotropin in CaSki cells: a new epidermoid cervical cancer cell line. Science 196: 1456-1458, 1977 PubMed: 77195470 Baker CC, et al. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61: 962-971, 1987 PubMed: 87141358 Pater MM and Pater A. Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 145: 313-318, 1985 PubMed: 85274378 Yee C, et al. Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am. J. Pathol. 119: 361-366, 1985 PubMed: 85248811 Hendricks DT, et al. FHIT gene expression in human ovarian, endometrial, and cervical cancer cell lines. Cancer Res. 57: 2112-2115, 1997 PubMed: 97330646 Hay RJ, Caputo JL, and Macy ML, Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC. Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988. Fleming DO, Richardson JH, Tulis JJ and Vesley D. (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC. Centers for Disease Control (1993), Biosafety in Microbiological and Biomedical Laboratories Human Health Service Publication No. (CDC) 93-8395. U.S. Dept. of Health and Human Services; 3rd Edition U.S. Government Printing Office Washington D.C. ATCC WARRANTY The viability of ATCC products is warranted for 30 days from the date of shipment. If you feel there is a problem with this product, contact Technical Services by phone at 800-638-6597 or 703-365-2700 or by e-mail at tech@atcc.org. Or you may contact your local distributor. DISCLAIMERS This product is intended for laboratory research purposes only. It is not intended for use in humans. While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, and use. ATCC is not liable for any damages or injuries arising from receipt and/or use of this product. While reasonable effort is made to insure authenticity and reliability of strains on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of cultures. Please see the enclosed Material Transfer Agreement (MTA) for further details regarding the use of this product. The MTA is also available on our Web site at www.atcc.org. © ATCC 2009. All rights reserved. ATCC® is a registered trademark of the American Type Culture Collection. 05/09 Cell Line Description References (additional references may be available in the catalog description at www.atcc.org) Please see the enclosed Material Transfer Agreement (MTA) for further details regarding the use of this product. The MTA is also available on our Web site at www.atcc.org.