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Quality Control: Verified to be free of bacterial and fungal contamination; free of mycoplasma by PCR assay; free of contamination with marmoset cells by isoenzyme electrophoresis for glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and nucleoside phosphylase.

The current lot of EBV virus has been shown to transform most B-lymphocytes within 3 to 4 weeks. The efficacy of this lot of virus has been compared to previous lots of EBV virus prepared by Coriell Cell Repositories in terms of the cell count at critical stages in the transformation process and in the time necessary for achieving a stable transformation.

Product Number: S016291
Lot Number: F1029
Volume per aliquot: 1 ml
Date Lot prepared: 10/6/11
Date Lot passed QC: 10/19/11

Storage of EBV supernatant: It is recommended that the frozen vial be stored at -70C or lower. Thawed virus preparation may be kept at 4C for up to 6 months without loss of transformation potential.

Safety Precautions for Glass Ampules: If the ampule is placed in liquid nitrogen upon receipt, special safety precautions should be followed when removing the ampule from storage. The potential exists for glass ampules to shatter when undergoing rapid temperature change after storage in liquid nitrogen. Protective gloves and clothing should be used and a face shield must be worn when thawing the ampule.

Glass ampules of EBV should be thawed by rapid agitation in a 37C water bath. The ampule should be wiped with 70% ETOH and the neck scored with a sharp needle file that has been immersed in 70% ETOH. Applying pressure with the thumbs (protected by a special holder or several layers of sterile gauze) on the side opposite the nick should produce a clean break of the neck of the ampule. Transfer the contents of the ampule using sterile technique.

Protocol for Transforming B lymphocytes by EBV (abbreviated):

1. Collect 5-10 ml of peripheral human blood in collection tubes containing either acid-citrate dextrose or K-EDTA as an anticoagulant.
2. Separate peripheral blood mononuclear cells (PBMC) by centrifugation on a Ficoll gradient.
3. Wash PBMC two times with RPMI-1640 with 25mM HEPES, pH 7.4.
4. Resuspend PBMC in a volume of medium equal to the starting volume of blood (RPMI 1640 with 20% FBS, 2mM L-glutamine or eqivalent, 2.0 g/L glucose, 2.0 g/L sodium bicarbonate and no antibiotics) in a T25 tissue culture flask.
5. Add 1 ml EBV and phytohemaglutinin to a final concentration of 10 µg/ml. Incubate at 37C, 5% CO2.
6. Twice weekly, examine flask for a change to an acidic pH and the appearance of "clumps" of cells growing in suspension. Adjust the volume of medium in the flask by removing spent medium and adding more or less fresh medium to maintain a slightly acidic pH. Be sure to let the cells settle to the bottom of the flask before adjusting the volume of the medium.
7. By 21 to 35 days in culture, the volume should have increased to approximately 20 mls. Cells should be growing in loose aggregates that can be broken apart by gentle trituration. At this time the lymphocytes can be subcultured at a seeding density of not less than 2x10⁵ viable cells per ml.
8. When cell density reaches 8x105 to 1x10⁶ cells per ml, the culture should be split at not less than 2x10⁵ cells per ml or cell stocks should be cryopreserved.
9. See the CCR Website at (Lymphoblast Cell Culture FAQ) for additional information about lymphoblast culture.

Biosafety Level 2: This product is to be handled as a potentially biohazardous material under Biosafety Level 2 containment or higher. Information is available in the U.S. Government Publication Biosafety in Microbiological and Biomedical Laboratories, Centers for Disease Control (1999)(www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm).

References:
Bolton, BJ. Spurr, NK. B-Lymphocytes. In Freshney, RI and Freshney, MG (ed): Culture of Immortalized Cells; Wiley-Liss, New York, 1996, pp 283-297.
Tumilowicz JJ. Gallick GE. East JL. Pathak S. Trentin JJ. Arlinghaus RB. Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources. In Vitro 20: 486-492, 1984. PubMed: 6086497
Miller G. Shope T. Lisco H. Stitt D. Lipman M. Epstein-Barr virus: transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes. Proc. Natl. Acad. Sci. USA 69: 383-387, 1972. PubMed: 4333982

Use Restrictions: NO WARRANTIES, IMPLIED OR EXPRESSED, ARE MADE REGARDING THE USE OF THIS MATERIAL TO EFFECT TRANSFORMATION OF B LYMPHOCYTES OR FOR ANY OTHER PURPOSE. The purchaser accepts all responsibility for complying with appropriate safety precautions. Please review the suggested minimum safety guidelines.