B-cell Immortalization Services

A laboratory photo used to illustrate b cell protocols.General Protocol for the Immortalization Of Human B-Lymphocytes Using EBV

To Submit Samples for B-cell Immortalization

The TCF can isolate lymphocytes from your blood samples and establish immortal (continuous) B-cell lines (lymphoblasts). Lymphocytes are isolated from whole blood by gradient centrifugation with Ficoll and transformations are initiated by Epstein-Barr virus infection. Epstein-Barr virus (EBV) is one of the herpes viruses and is harvested from cultures of the marmoset cell line B95-8. Human B-cells have a receptor for EBV and once infected, human B-lymphocytes can become immortalized with a high rate of success. They can then be propagated indefinitely in culture for experiments or cryopreserved for future needs.

The TCF will transform fresh or frozen lymphocytes using our protocol with a success rate that generally exceeds 98%. Blood samples should be collected in heparinizedtubes (green top vacutainers) and we recommend using a 21 1/2 guage needle to decrease cell lysis. Samples should be submitted within 24 hours of collection and kept at room temperature. (Frozen blood samples are acceptable, but the success rate will be dependent upon cell viability after the freeze/thaw process.) A volume of 20 ml of whole blood is recommended or a minimum of 2 x 106 fresh lymphocytes or 5 x 106 frozen lymphocytes if you have already separated the cells. Please call ahead if you are interested in this service so that we may prepare reagents and schedule staff.

What you get:
1 x 25 sq cm flask of immortalized cells in mid-log growth phase
2 - 5 x 1ml vial (depending upon yield) of frozen non-transformed lymphocytes for back-up/reference

Typical delivery time:
4 to 6 weeks

$125 per sample

EBV Stocks for Immortalizing B-cells in Your Laboratory

The TCF will make limited quantities of the stock preparations we use for B-cell immortalization available to LCCC member labs for a nominal charge ($30.00 per 1 ml vial). These stocks will have been screened for mycoplasma and will be tested for efficacy, however, there will be no warranty, expressed or implied, with regard to your success in immortalizing cells with these stocks. We strongly urge you to have the TCF staff perform this service for you. The protocol that we use and the familiarity of the entire process by the staff will increase the probability of successfully immortalizing your samples at a cost very close to or possibly less than it would cost you or your lab personnel to do it.