Study on Seminal Attributes of X- sperm Enriched Sahiwal Bull Semen

The present study was conducted to observe the effect of percoll density gradient centrifugation on quality of semen. Ejaculates were collected by AV method from Sahiwal bulls. X-sperm enrichment was done by percoll density gradient method i.e. 7 layers (70-10%). Centrifugation was done at 750 g (22-24°C) for 15 min. The pellets obtained were diluted in EYC medium. Semen quality was evaluated in fresh semen (Control), in pellet of normal centrifugation (Group I), supernatant of centrifugation in percoll density gradient (Group II) and pellet of centrifugation in percoll density gradient (Group III). To assess the quality of enriched semen pH, mass motility, progressive motility, live spermatozoa %, abnormal spermatozoa %, HOST % and intact acrosome % were evaluated. Number of progressively motile sperms in pellet of X- enriched semen were non-significantly increased and significantly (P<0.05) decreased in supernatant. The abnormal spermatozoa (%) were decreased in G III as compared to G II Live spermatozoa (%) were increased in enriched semen (pellet). Number of Intact sperms decreased significantly (P<0.05) in supernatant of percoll density gradient centrifuged Sahiwal semen. HOST responsive sperms number was not affected after percoll density gradient centrifugation. Thus, the semen quality of X-sperm enriched semen by percoll density gradient method (7 layer 70%) was not affected hence it can be used to increase female calves’ birth after A.I.

Correlation between adhesion molecules on the surface of red blood cell membrane and red blood cell lifespan / 中国输血杂志

【Objective】 To explore the correlation between red blood cell lifespan and adhesion molecules on the surface of red blood cell membrane, in order to establish a method to detect the duration of red blood cell storage. 【Methods】 10 samples(10 mL each) of fresh red blood cell, collectedf rom 10 healthy voluntary blood donors, were divided into 5 age groups (layers) by Percoll density gradient centrifugation. The expression of CD47, CD44 and CD147 on the surface of red blood cell membrane in each layer was detected using flow cytometry. The variance of protein expression in each layer of red blood cells was analyzed by SPSS statistical software. 【Results】 The expression levels (%) of 3 adhesion molecules on the surface of red blood cell membranes from young to old were CD47: 14.44±2.61, 9.30±1.75, 7.84±1.49, 6.54±1.32 and 5.53±1.12 (P<0.01); CD44: 25.01±1.94, 19.22±1.52, 17.10±1.28, 15.18±1.11 and 13.56±1.08 (P<0.01); CD147: 33.46±1.99, 28.31±2.95, 23.83±1.59, 20.40±1.56 and 18.03±1.65 (P<0.01). 【Conclusion】 The expression levels of CD47, CD44 and CD147 on the surface of red blood cell membranes have showed a downward trend as the storage extended. These three protein adhesion molecules have showed a correlation with red blood cells lifespan, and could be used as detection markers of cell age.

A comparative study of tumor-associated macrophages isolation in colorectal carcinoma by Ficoll-Hy-paque density gradient centrifugation and Percoll density gradient centrifugation / 实用医学杂志

Objective Percoll density gradient centrifugation and Ficoll-Hypaque density gradient cen-trifugation, which are frequently-used methods for separation of tumor-associated macrophages (TAMs) from solid carcinoma were compared, in order to find an effective way to separate TAMs from colorectal carcinoma (CRC). Furthermore, we studied the best adherence time of separating macrophage among mononuclear cells. Methods specimens were collected from CRC patients , after digesting into single cells , TAMs were separated from the same specimen by 100% Ficoll, 35% percoll and 25% combined with 65% percoll respectively. After these pre-liminary separation, the collected cells were purified a second time by adherence separation. The purity of TAMs were detected by immunofluorescence. Results TAMs purity from Ficoll-Hypaque density gradient centrifugation was 80.18%, statistically higher than that from Percoll density gradient centrifugations (54.33% and 10.93% re-spectively). Conclusion Compared to Percoll density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation is a more effective and simple way to isolate TAMs from colorectal carcinoma , suggesting it can be wildly used in clinical and basic medical research. 2-4 hours is the best adherence time for isolating macrophage.

Effects of IGF (Insulin-like growth factor) on sub-populated articular chondrocytes by Percoll density gradient / 대한정형외과연구학회지

PURPOSE: Articular chondrocytes have been known to have heterogeneity in articular cartilage. The different responses of chondrocytes to various cytokines and growth factors have been reported. These variations are likely a result of metabolic differences among the cell populations. We used the Percoll density gradient method to separate chondrocytes from articular cartilage into distinct subpopulations. Several growth factors are known to enhance the synthesis of cartilage matrix. In particular, IGF has specific anabolic effects. Addition of IGF to chondrocytes increased the synthesis of proteoglycans and collagen type-II while inhibiting the degradation and release of proteoglycans. MATERIALS AND METHODS: Chondrocytes were isolated from rabbit knee articular cartilage by collagenase digestion. In brief, male rabbits weighing 250g were euthanized by injecting an overdose of Nembutal, and nonfibrillated articular cartilage of the knee was removed by sterile dissection. Isotonic Percoll was mixed with 10x PBS to give a 60% stock solution. This was further diluted with PBS to give Percoll concentrations of 10, 20, 30, 40, 50, and 60%. RT-PCR, western blot analysis, immunocytochemistry, and immunohistochemistry were done for examination of collagen type II and aggrecan as the specific marker of extracellular matrix and proteoglycan synthesis on cultured chondrocytes. RESULTS: The sub-populated cells were proliferated variously. On the other hand, the addition of IGF to the sub-populated cells increased the proliferation in all fractions. Also the expression of collagen type-II and TIMP-2 was increased by IGF treatment. After alginate culture, collagen type-II expression was not significantly different between the IGF treated and the control groups in high density fractions. However, the addition of IGF to chondrocytes increased the expression of collagen type-II in low density fractions. The expression of collagen type-II after IGF addition was decreased in monolayer culture while it was increased in alginate culture. CONCLUSION: The effects of IGF are various among the subpopulated chondrocytes. These results will provide useful information for the separation of articular chondrocytes with an active metabolic activity and extracellular matrix for the investigation of the pathogenesis of articular cartilage.

Subpopulation of Rabbit Articular Chondrocytes Separated by Percoll Density Gradient / 대한정형외과연구학회지

Articular chondrocytes have been known to have heterogeneity in articular cartilage. Four layers are generally recognized from the articular surface to the subchondral bone. We have used Percoll density gradients to separate chondrocytes from articular cartilage into distinct subpopulations. Non-fibrillated articular cartilage was obtained from rabbit knee. The cells were carefully layered on the top of the preformed gradient and spun. After centrifugation, we obtained four fractions: Fraction A referred boundary between 0% and 10%, fraction B from between 10% and 20%, fraction C from between 20% and 30%, and fraction D from between 40% and 50%. In the A fraction, cells are relatively larger and round in shape, while their nuclei are relatively smaller. In the cytoplasm many lipid droplets were found and rough endoplasmic reticulum were disrupted. In the D fraction, chondrocyte is small, with large nucleus which surrounded by well-developed rough endoplasmic reticulum. The type II collagen proteins were expressed strongly and more proteoglycans synthesized in fractions A and B. And chondrocytes from the fraction D divided more slowly than those from the fractions A, B, and C. We have succeeded in separating chondrocytes from articular cartilage into distinct subpopulations by Percoll density gradients, as well as characterized growth rate, histological appearances and phenotypic expression. This study is the first report about the Percoll density gradients to separate articular chondrocytes.