Cell Culture Services

Production and Purification of Antibodies from Hybridoma Lines

We produce and purify antibodies from culture supernatants or ascites. Hybridoma cells can be grown either in roller bottles or in hollow fiber cartridges. The culture supernatant (1 liter or more) is precipitated by ammonium sulphate and IgG is purified by protein G chromatography through FPLC.

Antibody Screening by ELISA

Although we are not raising monoclonal antibodies any more, we screen supernatants by ELISA for subcloning of positive clones and other purposes.

Mycoplasma Testing

IMPORTANT:

Culture your cells at least two days without any antibiotics (without PennStrep, G418, etc.), prior to removing a sample cell culture supernatant for testing. Most common strains of mycoplasma grow slowly in the presence of antibiotic and may be present only in spurious amounts and thus may be undetectable. Testing cells grown with antibiotics often gives false negative results because most mycoplsama starins are dormant but not dead in the presence of antibiotics.

Since infected cells secrete mycoplasma into the medium, testing the cell culture supernatants is sufficient to detect mycoplasma infection.

Detection of Mycoplasma by MycoAlert (Cambrex)

This kit is very simple to use, extremely rapid, highly sensitive and is generic for all common mycoplasma species. Mycoplasma have unique metabolic processes that derive energy from their host cells. The method specifically detects enzymes involved in mycoplasma metabolism that are absent in host cells. By using substrates for the mycoplasma specific enzymes, ADP is converted to ATP. The resultant ATP is conveniently detected using the bioluminescent reaction catalyzed by the Firefly Luciferase enzyme. Any increase in light output from this reaction, as measured by a luminometer, will indicate the presence of mycoplasma.

Duration is usually 1 week. Please note that samples should arrive at our facility by Wednesday afternoon before 4 pm.

Please contact Sabine Baxter (215-898-2795) for details.

Endotoxin Testing

We test water or samples of media to determine the level of endotoxin using the LAL (Lumilius Amebosite Lysate) gel clot assay from Associates of Cape Cod, MA. At least 200 µl samples should be submitted in endotoxin free container. Generally radiation sterilized tissue culture plasticware is endotoxin free, but not autoclaved glassware. Three dilutions per sample are tested routinely, along with a set of blanks and positive controls.

EBV Transformation

Epstein Barr Virus (EBV) transformation is a reliable method to immortalize mammalian cells. It is most often used to obtain cell lines from human lymphocytes that serve as a permanent source for DNA isolation and protein isolation. The technique has found widespread use in clinical trials as the principal method of generating a permanent source of patient DNA for genotyping. Patient genotyping is becoming more popular as the genetic causes of diseases are being unraveled at increasing speed.

EBV is the causative agent of human infectious mononucleosis , a childhood disease. More than 90% of adults are a carrier of EBV. Adults usually show no EBV symptoms.

EBV is a Herpesvirus and its genome consists of a 172kb linear double stranded DNA which has been completely sequenced. EBV molecular biology and pathogenesis are extensively studied and the roles of many crucial EBV and host cell genes in pathogenesis are known. EBV infects only certain mammalian epithelial cells and B lymphocytes. In vitro EBV immortalizes B-cells by activating a number of cell cycle regulating genes as well as B-cell specific genes including immunoglobulin genes. Usually B-cell infections are latent and only strong stimulation can cause the lytic cycle. EBV DNA is replicated as an episomal ring and 10-20 copies per cell are typical.

IMPORTANT: When working with EBV (or any other infectious agent) negative control blood samples may NOT be obtained from laboratory personnel in order to minimize the risk of infections with one's own EBV-transformed blood cells against which the body would not be able to mount an immune response.

Please contact Leota Terri (215-898-2795) for details.

Special Media

The facility prepares and sterilizes custom formulations as long as a commercial powder base is available as a reasonable starting point.

Fly Food

The facility prepares media for Drosophila culture in vials and bottles using the method of Spradling. We also make a modified version where the molasses is replaced by dextrose.