Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome

Abstract

BackgroundThe effects of fetal calf serum (FCS) heat inactivation and bacterial lipopolysaccharide (LPS) contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM) cells were grown in FCS, either non-heated, or heat inactivated, having low (< 1 EU/mL) or regular (< 30 EU/mL) LPS concentrations. Protein lysates were resolved by 2-DE followed by phospho-specific and silver nitrate staining. Differentially regulated spots were identified by nano LC ESI Q-TOF MS/MS analysis.ResultsA total of four proteins (EIF3M, PRS7, PSB4, and SNAPA) were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE) as compared to cells grown in media with non-heated FCS (NHE). Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN) displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1).ConclusionThe present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.

Probing the prostate tumour microenvironment I: impact of glucose deprivation on a cell model of prostate cancer progression

“…Hence, the carefully selected five fold reduction in the concentration of glucose used in these in vitro experiments can be considered reflective of changes encountered by the prostate tumour cells in vivo (13). Similarly, FCS was not omitted from the culture media when generating a low glucose microenvironment so as to avoid changes induced by restricting the proliferative capacity of the cells and potentially inducing apoptosis in a manner that varied between the cell lines [ 16 ]. Therefore, by undertaking the experiments in the presence of FCS, the observed proteome-wide alterations in protein expression are more likely to be solely a consequence of the change in glucose concentration.…”

Toxoplasma gondii: Prolonged in-vitro maintenance of virulent tachyzoites in fluid media at low temperatures

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“…7 On the contrary with this, the presence of FBS in the media has several drawbacks, such as it contains several complement proteins that have a principal role in innate immunity influencing the results of immunoassays when added to culture cell lines. 15 FBS contains a diverse repertoire of protein-coding and regulatory RNA species; the majority of them are retained even after extended ultracentrifugation. 17 FBS contains some inhibitory factors such as endotoxins as well as the specific or cross-reacted antibodies that form a problem on the level of purification of the produced T.g.T.…”

Postnatal ex vivo rat model for longitudinal bone growth investigations

“…Slides were stained with 0.1% toluidine blue O, in PBS at pH 5.6, and von Kossa stain (Acros Organics, New Jersey, USA). The FBS used in this study was inactivated as described by Rahman et al . The inactivation was achieved using gradual heating in a water bath at 56 ° C for 30 minutes prior to incorporation into the α‐MEM media.…”