Serum-free Media For Low Background and High Signal in T Cell and B Cell Assays ven carefully selected serum batches are one-of-a-kind reagents. Unique levels of bioactive molecules in different serum batches affect the baseline activation level (background) and the magnitude of antigen-induced T cell and B cell activation (signal). The CTL Serum-free Media portfolio was developed for low background and high signal to outperform the best sera, and are qualitycontrolled for optimal and consistent performance in T cell and B cell assays. Low Background Low background, or ideally no background at all, is critical for PBMC-based T cell assays because rare antigenspecific T cells need to be detected amongst all other cells. The frequency of antigen-specific T cells seldom reaches one within a thousand PBMC, and often is as low as one within a hundred thousand, and less. Only in low-background assays can such low-frequency T cells be reliably detected. Each batch of CTL Serum-free Media is quality controlled for low-background activity, typically providing zero to five nonspecific IFN-γ, IL-2, IL-4, IL-5 and IL-17 ELISPOTs in PBMC of healthy donors. Serum, in contrast, frequently causes an elevated background (Figure 1A). Even brief exposure of PBMC to serum with mitogenic properties, e.g., during cryopreservation, or when washing the IFN-γ Spots / 400,000 PBMC Primary cells need the multitude of growth and nutrition factors contained within serum to survive and function. T cells and antigen-presenting cells (APC) are particularly vulnerable and dependent on these factors when freshly isolated from blood, tested in vitro, cryopreserved, or thawed. In the past, carefully selected sera were needed to support PBMC during isolation, testing, freezing, and thawing. No longer! CTL-Test™ Serum Figure 1: Performance of serum-free CTL Test™ Medium compared with eight different qualified sera. 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 (SI: Antigeninduced 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). cells, can result in a high background. Entire T cell monitoring trials have been lost because PBMC were either frozen or tested with mitogenic serum! High Signal Serum always contains suppressive factors, such as TGFβ and IL-10. If present in increased concentrations, such molecules inhibit T cell activation, jeopardizing the detection of antigenspecific T cells in functional assays. For detecting antigen-specific CD8 cells, CTL Serum-free Medium matches or outperforms antigen-induced ELISPOT counts elicited with sera that have been qualified for T cell assays (Figure 1B). For CD4 cell detecti

Serum-free Media Standardization and Quality Control Using different sera while working with PBMC is likely to produce different results. Each serum is a unique reagent containing unique concentrations of molecules that activate or inhibit T cells and APC. The need to work with serum has been a major obstacle for standardized and reproducible ex vivo experimentation. No longer! CTL Serum-free Media contain highly-defined and constant bioactive components, and each batch is quality controlled for consistant performance. The CTL Serum-free Media platform permits assay standardization within a laboratory,...