Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth. Quality control tools for FBS are relatively primitive and expensive given the complexity of the sample and the large amounts of FBS used. We undertook this study to examine whether proteomics could be used as a tool to analyze the variability of different fermentation processes. We hypothesized that inconsistent cell growth in fermentations could be due to the quality of FBS and that different lots of FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. To investigate whether this was the case, we grew three batches of adult retinal pigment epithelial cells (ARPE-19) using three different lots of fetal bovine serum (FBS-Ia, FBS-Ib, and FBS-II). We found that the growth rate of the culture was significantly and consistently higher in the FBS-II lot. To determine why the other lots promoted different growth properties, we used proteomic techniques to analyze the protein composition of the three lots. We then performed a time course study to monitor specific changes in individual proteins in the fermentation medium. The amount of several extracellular matrix and structural proteins, which are indicators of cell growth, increased over time. Alternatively, components supplied by the FBS addition, such as nutritional-related and cell-spreading-related proteins, decreased over time.

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Infection of epithelial cells by two biovars of Chlamydia trachomatis results in the tyrosine phosphorylation of several host proteins. The most prominent change in host protein tyrosine phosphorylation involves a complex of proteins with molecular masses of 75 to 85 kDa (pp75/85) and 100 kDa (pp100). The C. trachomatisinduced tyrosine phosphorylation of pp75/85 and pp100 is observed in several cell lines, including epithelial cells, fibroblasts, and macrophages. Subcellular fractionation and detergent solubility properties of pp75/85 are consistent with its association with the cytoskeleton. Phosphoamino acid analysis demonstrates that the pp75/85 complex is phosphorylated on both tyrosine and serine residues. Immunofluorescence studies of chlamydia-infected cells by using fluorescein isothiocyanate-phalloidin and antibodies to phosphotyrosine and cortactin demonstrate that tyrosine-phosphorylated proteins, as well as cortactin, are localized to the chlamydial vacuole and that this process is facilitated by actin. MATERIALS AND METHODS Cell culture. The human epithelial HeLa cell line (ATCC CCL2; American Type Culture Collection, Rockville, Md.) was grown in Dulbecco's modified essential medium H16 (DME-H16 medium) supplemented with 5% (vol/vol) fetal calf serum (FCS) (DME-5). The mouse fibroblast L929 cell line (ATCC CCL1) was grown in RPMI 1640 medium containing 25 mM HEPES and 10% (vol/vol) FCS. The murine macrophage cell lines RAW 264.7 (ATCC TIB 71) and J774A.1 (ATCC TIB 67) were cultured in DME-H21 medium supplemented with 5% (vol/vol) calf serum and 5% (vol/vol) FCS. All cell lines were grown at 37°C in 5% CO 2. Medium was obtained from the Cell Culture Facility, University of California, San Francisco. FCS was obtained from Gibco (Bethesda, Md.). Growth and purification of chlamydiae. The MoPn biovar of C. trachomatis was routinely grown in HeLa cells for 48 h, after which it was purified as described previously (10). The LGV L2 serovar was obtained from Richard S. Stephens (University of California, Berkeley) and propagated in L929 cells. In some experiments, the MoPn biovar was purified by centrifugation on a discontinuous Renografin (Squibb Diagnostics, BMS Pharmaceutical Group, Irvine, Calif.) gradient (17). Infections were carried out at a multiplicity of infection (MOI) of 20 to 100 inclusion-forming units. Despite the high-MOI inocula used, no immediate cytotoxicity (prior to 18 h) was observed. Chlamydial infection and protein extraction. Subconfluent monolayers of HeLa, L929, RAW, or J774 cells (2 ϫ 10 5 cells) in 100-mm-diameter plates were infected for 1 h at 37°C with a purified preparation of MoPn or LGV in sucrosephosphate-glutamine buffer (10) diluted in DME-5. The inoculum was removed and replaced by fresh medium, and the infection was allowed to proceed for different periods of time. For mock infections, the cells were incubated with a preparation of uninfected cells purified by the protocol used for chlamydiae. After the medium was removed, the cells were washed with and scraped into cold

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