Stable Cell Line Protocol

The first critical step for stable.

Stable cell line protocol.

Split confluent cells 1 5 in 10 ml dmem 10 fbs media. Use the lowest concentration of drug that begins to give massive cell death in 3 days and kills all the cells within two weeks. Transfect cells with desired plasmid construct s. Store at 4 c.

Our xtenchotm cell line together with our in house xten protocol is your best chance at overcoming your difficult to express protein challenge. Select and expand stable. While performing the kill curve 1 week optimize transfection. 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.

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. 10 v v fbs and 4mm l alanyl l glutamine. Protocol of stable cell line generation 1. For transfection please follow the manufacturer s instruction of your transfection system.

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 protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells. Stable cell line generation protocol generate a kill curve to determine the optimal selection antibiotic concentration. Incubate the cells for 10 days replacing selective medium every 4 days or as needed.

The following day replace the standard media with media containing g418 g418 geneticin is an aminoglycoside antibiotic similar in structure to gentamicin b1. Introduction this protocol describes the procedure for creating a stable cell line based on the expicho expression system for use in commercial bioproduction. Choose the g418 concentration. There are many different approaches for establishing stable cell lines depending on the type of expression you re interested in inducible vs.

Transfer 0 5 ml cell suspension into 24 well plate containing 500 µl of media drug. This protocol is specific for the establishment of cell lines that constitutively express gfp tagged proteins. 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. Expicho s cells are transfected cloned and selected in expicho expression medium followed by the scale up in the bioproduction amenable expicho stable production medium.