The CTL Serum-free Media portfolio was developed for low background and high signal to outperform the best sera, and are quality-controlled for optimal and consistent performance in T cell and B cell assays.
CTL-Cryo™ ABC Media Kit
Serum-free freezing media kit for cryopreservation of freshly-isolated PBMCLearn more
Performance of Serum-free CTL Test™ Medium Compared with Eight Difference Qualified Sera
Individual healthy donor cells are available in batches of up to 1200 vials for any application that requires live, functional PBMC.
Cryopreserved PBMC of the same batch were thawed and tested in an IFN-γ ELISPOT assay in eight different laboratories using either CTL-Test™ (blue), or, in parallel in the serum that the respective laboratory has selected for T cell work (green). Spot counts obtained in the medium control are shown in Panel A. The antigen- (CEF peptide-) induced spot counts are shown in Panel B. The stimulation index (St: antigen-induced spots/medium background) defining the strength of signal measured is also shown for each of the laboratories for the results obtained with CTL-Test™ (SI: CTL- Test™), and with the respective laboratory's serum (SI: Serum). In all cases, the maximal spot counts with CTL-Test™ were equal to better than the sera, and four of the eight sera induced an elevated background (Zhang et al., J. immunotoxicology, 2009, 6:227).
CTL Serum-free Media Outperforms Serum-containing Media in Most Instances
IFN-γ ELISPOT assay performance with serum-containing complete RPMI medium vs. the Serum-free CTL-Test™ Medium. Freshly-isolated PBMC of three healthy donors were tested in parallel under both conditions; the number of spots obtained (Y axis) or in serum (X axis) after stimulation with antigens that stimulate CD4 cells, Mumps, PPD and Candida, is represented by the dots. The red line indicates equal perfor- mance. The medium background was zero for all conditions. Note, several recall responses that were not detectable in serum became clearly positive in CTL Test™ Medium.
Scientific Background and Utility of Serum-free Media in ELISPOT
Serum-free cell culture media allow users to standardize their cell culture conditions by avoiding the use of undefined and highly-variable serum products derived from humans or animals, e.g. human AB serum or fetal calf serum (FCS).
The high variability in the biological properties of different serum batches makes it necessary to pre-screen many batches in order to obtain a batch that is well-suited for a given application. Even a brief exposure of PBMC to a mitogenic serum batch, for example during washing or freezing of these cells, will result in a high background in cytokine assays. In addition, toxic or inhibitory serum batches will jeopardize the assay results. The unique performance of each serum batch necessitates the purchase of large lots to assure consistency of assay conditions as long as possible. This adds to the inconvenience and cost associated with the use of serum. Switching to or using a different batch of serum introduces a significant unpredictable variable to the test conditions each time, making test results difficult to compare. Furthermore, infectious risks associated with serum are leading to increasingly tighter restrictions for international shipments and exchange of any material that has been exposed to serum.
For all these reasons, there is considerable pressure from regulatory agencies and from the scientific community to avoid the use of serum and move to consistent, defined substitutes. However, because serum is naturally rich in a multitude of growth factors and other essentials for cell growth and functionality, it has been challenging to develop a serum substitute for primary cells in general, and for PBMC in particular. Most serum-free media contain enough additives to permit the growth of robust tumor cells, and hybridomas, but freshly-isolated human PBMC require more stringent conditions to maintain their viability and functionality in serum-free media.
CTL has been working for years with US governmental, industrial, and academic partners to standardize ELISPOT and other direct ex vivo cytokine assays performed on freshly-isolated or cryopreserved PBMC. These efforts have resulted in the CTL Serum-free Media Platform, which consists of three product lines that have been carefully designed to cover the four critical steps of working with human PBMC: washing, testing, freezing, and thawing. All four previously required the use of serum.