Stable Cell Line Protocol

This protocol is specific for the establishment of cell lines that constitutively express gfp tagged proteins.

Stable cell line protocol.

Constitutive and the construct that you are incorporating. Transfect cells with desired plasmid construct s. Susceptibility to g418 is different among cell lines which many even vary with cell. Store at 4 c.

Split a confluent dish of cells at approximately 1 5 to 1 10 depending on the cell type and cell density post transfection into medium containing various concentrations of the antibiotic. This is usually the first step before moving to stable cell line development in order to be able to ensure that all antibody features meet the requirements in cho based productions. The following day replace the standard media with media containing g418 g418 geneticin is an aminoglycoside antibiotic similar in structure to gentamicin b1. 10 v v fbs and 4mm l alanyl l glutamine.

Protocol of stable cell line generation 1. Choose the g418 concentration. Transducing cells with lentivirus results in a heterogenous polyclonal population that varies in the number of integration events and the site s of proviral integration across cells. The first critical step for stable.

Stable cell line generation protocol generate a kill curve to determine the optimal selection antibiotic concentration. Transfer 0 5 ml cell suspension into 24 well plate containing 500 µl of media drug. Use the lowest concentration of drug that begins to give massive cell death in 3 days and kills all the cells within two weeks. Select and expand stable.

To a 500 ml bottle of dmem high glucose add 55 ml of heat inactivated fbs and 11 ml of 200 mm l alanyl l glutamine. Incubate the cells for 10 days replacing selective medium every 4 days or as needed. Introduction this protocol describes the procedure for creating a stable cell line based on the expicho expression system for use in commercial bioproduction. Our xtenchotm cell line together with our in house xten protocol is your best chance at overcoming your difficult to express protein challenge.

To set up for the stable cell line selection split cells and transfect with your construct. This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells. While performing the kill curve 1 week optimize transfection. Split confluent cells 1 5 in 10 ml dmem 10 fbs media.