科研之友
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摘要
A modified procedure of Percoll density gradient centrifugation was developed to isolate and fractionate synchronous cells from stationary phase (sp) cultures of different yeast strains, as well as Raman spectra discrimination of single yeast cells was reported. About 1. 75 mL Percoll solution in 2 mL polypropylene centrifugal tube was centrifuged at 19 320 g, 20 degrees C with an angle rotor for 15 min to form continuous densities gradient (1. 00 similar to 1. 31 g . mL(-1)), approximately 100 mu L sample was overlaid onto the preformed continuous density gradient carefully, subsequently, centrifuged at 400 g for 60 min in a tabletop centrifuge equipped with a angle rotor at 25 degrees C. Yeast samples could be observed that the suspensions were separated into two cell fractions obviously. Both fractions of different yeast strains were respectively determined by differential interference contrast (DIC), phase contrast microscope and synchronous culture to distinguish their morphological and growth trait. The results showed that the lower fraction cells were unbudded, mostly unicellular, highly refractive, homogeneous and uniform in size, and represented growth characteristic synchronously; Their protoplasm had relatively high density, and contained significant concentrations of glycogen; all of which were accordant with description of quiescent yeast cells and G(0) cells in previously published paper. It was shown that lower fraction was quiescent cells, synchronous G(0) cells as well. A Raman tweezers setup was used to investigate the differences between two fractions, G(0) cells and non G(0) cells, at a single cell level. The result showed that both G(0) cells and the non G(0) cells had the same characteristic peaks corresponding biological macromolecules including proteins, carbohydrates and nucleic acids, but all characteristic peak intensities of G(0) cells were higher than that of non G0 cells, implied that the macromolecular substance content of G(0) cells was more higher. Principal component analysis (PCA) was performed between G(0) cells and non G(0) cells, the results showed that the chemical composition content among the synchronization G(0) cells has less difference, and G(0) cells were homogeneous but non G(0) cells were heterogeneous, indicating single cell optical tweezers Raman spectroscopy could identify the synchronous and asynchronous cells. The modified method is feasible, economical and efficient highly. G(0) synchronous cells of most yeast strains could be isolated by a modification of Percoll density gradient centrifugation.
