之前已经介绍过了SCI中Discussion和Introduction的写法。接下来就说说Materials and Methods怎么写，这次主要是关于细胞实验方面的。大家直接当模板用就行啦。
说明：下文中，M=方法 A =病名 X=药名 C=细胞
药物稀释
X was diluted in a 0.1 M PBS (pH 7.4) to a concentration of 1.0 and 0.5 mg/ml.
分离获得的细胞数
Cell yields were related to the methods used for isolation. Both the M1 and M2 gave lower cell yields than the M3. After 4 hr of enzyme treatment, cell yields by the M1 and the M2 were 56.3% (P < 0.01, as compared with M3) and 68.0% (P < 0.05), respectively, of the yields obtained with the M3. The mean number of UM obtained from the iris was 1.57 ± 0.62 X 105 cells; from the ciliary body, 0.95 ± 0.35 X 105 cells; and from the choroid, 1.23 ± 0.66 X 106 cells. Together, approximately 1.5 X 106 UM cells could be isolated from one eye using the M3. Approximately 2.3X105 cells were isolated from each iris using this method, and the average viability was 90.2%; the growth rate was 68.2%.
细胞活性及生长
The viability and growth rate of X isolated by the M1 (88.1 ± 2.1% and 51.6 ± 1.6%, respectively) were lower than that of the M3 (91.1 ± 0.95%, and 59.8 ± 2.4%, P < 0.05). The viability and growth rate of X isolated with the M2 did not differ significantly from that of the M3. Most plated X attached and spread. Spreading occurred 1-3 d after plating. Using the M, X plating efficiency and spreading rate were 82.0 ± 1.80% and 65.8 ± 2.21%, respectively. X that attached but did not spread usually detached with time or were lost at the time of subculture. After 6-12 d of incubation, most of the spreading UM proliferated. A small part of X remained stationary, with dense pigmentation as compared to the diluted pigment in the dividing cells. Stationary cells persisted in the culture, but the proportion decreased with the growth of the dividing cells. The longest lived cell lines from iris and ciliary body have been passaged for 12 and 13 generations with 22 and 26 cumulative population doublings, respectively, and are still active. More than 80% of the cultures attained 8 or more cumulative population doublings. Cs grew well/ (slowly) in FIC medium, with a doubling time of 1.3 days/(with little increase in the number of cells after 5-7 days incubation). Cs cultured with X (at 3 M, on both 5th and 7th days) and X2 (at 10M on 5th day) showed a marked increase in the number of cells as compared with the negative controls. The number of cells incubated with the other PGs at all concentrations showed no significant difference when compared to the negative controls.
抑制生长
X cultured with--- medium grew much more slowly or did not grow/( ceased to grow), and showed a decrease of melanin content. X inhibited the growth and melanogenesis of UM cultured with complete medium.
促生长
Addition of X1, X2, and X3 at a concentration of 10-5 M resulted in significant stimulation of growth and an increase in the melanin content (P< 0.01). X stimulated cell growth significantly in cells cultured with --- medium and to a lesser degree in complete medium, but not in ---medium, indicating that X mainly activated the cAMP system. X stimulated growth and melanogenesis in a dose-dependent manner at concentrations ranging from --- to --- M. These results indicated that X only stimulated cell growth at a high concentration. These effects were dose-dependent at the range from --- to --- M
对生长无作用
X1, X2, and X3 at a concentration of 1mM showed no effect on cell growth or melanin content.
倒置相差显微镜下观察
X are easily identified and distinguished from other cell types based on these morphologic parameters by phase contrast microscopy. Morphologically, the X cells soon after isolation appeared as black spheroids, either as single cells or in small clusters. After attachment to the culture dish, most X cells spread into polygonal or cuboidal shapes; the cells had round nuclei and dense pigmentation. After subculturing, the X cells either remained polygonal or elongated to become spindle shaped, but they still expressed cytokeratin and S100 antigens. Morphologically, iridal melanocytes cultured with FIC medium displayed more dendritic processes (mean:2 cell−1) than those cultured with --- medium (2.06 cell−1).
透射电镜
Some of the cultures were studied by transmission electron microscopy. Cultured X displayed microvilli on the upper surface, and hemidesmosomelike structures were located at the bases of cells. Glycogen granules, mitochondria, rough endoplasmic reticulum, lysosomes, and lipofuscin were present in the cytoplasm. Round, mature melanosomes were also present in the cytoplasm, especially in the primary cultures and early passages.
免疫细胞化学染色
C stained positively with N1, N2, and N3 antibodies. C stained with N1and N2 but not with N3. C stained only with N1. X stained with anti-S100 protein and vimentin but not with anticytokeratin.
统计学
The differences of X and cell counts between the control and test groups were statistically significant (cell number: 0.05 > P>0.01 at concentrations of ---M in complete medium, at --- M in --- medium, and P< 0.01 at concentrations of --- M in --- medium). The differences between the X treated culture and negative controls were significant at concentrations of 10-6-10-5 M In the study on/(of the) cAMP-deleted medium, the number of cells incubated with X at all concentrations showed no significant difference compared with the negative controls.
