Abstract
ObjectiveCurrently, the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of the cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have a relatively equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 h of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry.ResultsCompared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.
Highlights
Sample collection Blood was collected from 32 healthy volunteers (Additional file 1: Table S1) in tubes containing EDTA-ACD
The findings introduce Autologous plasma (AP) as a better supplement for human neutrophil cell culture than fetal calf serum (FCS) and propose a simple and economical procedure for neutrophil isolation and culture
The rest of the cells were stained using an AnnexinV-FITC Apoptosis detection kit as per Neutrophil purity The granulocytes were located on forward scatter (FSC)/ side scatter (SSC) plots and suitable gates were set around them and around lymphocytes and monocytes
Summary
Neutrophil purity The granulocytes were located on forward scatter (FSC)/ side scatter (SSC) plots and suitable gates were set around them and around lymphocytes and monocytes. The corresponding SSC histograms were used to identify the number of cells in each gate. Neutrophil viability and apoptosis Neutrophils were stained by Annexin-V and propidium iodide (PI) to identify apoptotic and viable cells (Fig. 2). The viability of neutrophils in AP cultures and FCS cultures decreased over time. FCS group showed a more steep reduction in the viability (P = 0.003) and a meaningfully higher tendency to undergo apoptosis over the time (P < 0.05) (Fig. 3a, b). Oxidative burst rate and CD11b expression No significant differences were observed between AP and FCS cultures (Fig. 3c) (P = 0.632) in term of respiratory burst. Due to the substantial reduction in the number of neutrophils, it was not possible to assess the CD11b expression at 60 h. There were no significant differences between AP and FCS cultures (Fig. 3d). The data was further evaluated on the base of the gender of participates (Additional file 2: Figure S1)
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