Percoll Density Gradient Centrifugation Research Articles

Homologous transplantation of fluorescently labelled enriched buffalo (Bubalus bubalis) spermatogonial stem cells to prepubertal recipients

Spermatogonial stem cell transplantation provides a unique opportunity to study the biology of spermatogenesis and also offers an alternative approach for genetic modification in large animals. The present study aimed to extend this technique to the water buffalo. Spermatogonial stem cells (SSCs) were isolated from prepubertal buffalo testes (3-6 months of age) using two-step enzymatic digestion method and enriched by differential plating and Percoll density gradient centrifugation. The enriched SSCs expressed numerous spermatogonial transcriptional markers, viz. ID4, THY1, BCL6B, UCHL1, ETV5 and REX1 which confirmed their bonafide SSC identity. Subsequently, the enriched SSCs were labelled with a fluorescent dye PKH26 and transplanted into buffalo calves under ultrasound guidance. The recipient testes were recovered after 7-8 weeks by castration and their fluorescence microscopebased examination exhibited the persistence and localization of the fluorescent donor cells within the recipient seminiferous tubules. Further validation was done by the flow cytometric evaluation of PKH26 labeled donor cells among those isolated by two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated the feasibility of SSC transplantation technique in the water buffalo.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

An effective method of isolating microvascular endothelial cells from the human dermis.

Dermal microvascular endothelial cells (DMECs) play central roles in inflammation and angiogenesis and have become important cell models for studying various skin diseases. However, primary DMECs are difficult to culture and often contaminated by mesenchymal stem cells, fibroblasts, and other stromal cells. Surgically removed superfluous foreskin was first cut into pieces, digested with two types of enzymes, and dispersed into single cells. Cells obtained from the dermis were then subjected to Percoll density gradient centrifugation and cells located between densities 1.033 and 1.047 g/ml were further purified with endothelial growth medium containing decreasing concentrations of puromycin. Obtained HDMECs were identified by microscopy, flow cytometry, quantitative reverse-transcription polymerase chain reaction, western blot analysis, and immunofluorescent staining. The expression of CD31 (PECAM-1), CD34, VEGFR2, VWF (Von Willebrand Factor), VE-Cadherin (CD144), and NOS was positive. HDMECs were found to have abilities of angiogenesis and uptake of acetylated low-density lipoprotein. Growth curves and cell viability were analyzed, and a growth pattern consisting of the "latency phase-logarithmic growth phase-stagnation phase" was determined. In this study, a simple, rapid, effective, and low-cost method is established to isolate HDMECs from the foreskin with a purity of over 91% and high viability. The method showed good repeatability and allowed a stable passage. This study provides technical support and theoretical guidance for studying the physiological characteristics of HDMECs, the pathogenesis of the skin associated, and other microvascular diseases.

Classification and morphology of circulating haemocytes in the razor clam Sinonovacula constricta

The razor clam Sinonovacula constricta is widely distributed in the intertidal zones and estuarine waters along the coast of western Pacific Ocean and is extensively cultured. Even though haemocytes are known to play an important role in the immune mechanisms of bivalves, these cells are poorly studied in S. constricta. We researched the morphology and immunological activities of haemocytes in S. constricta using light and electron microscopy and flow cytometry. Three major subpopulations of haemocytes were identified in the haemolymph: granulocytes, semigranulocytes, and hyalinocytes. These subpopulations were divided using flow cytometry, but not satisfactorily. Therefore, the flow cytometry findings were combined with the light and electron microscopy findings, as well as Percoll density-gradient centrifugation findings, to classify and distinguish between the cell types more effectively. The combined findings showed that granulocytes was larger cells, while semigranulocytes was smaller and more abundant. Further, granulocytes had numerous granules in the cytoplasm, semigranulocytes contained fewer and smaller granules, and hyalinocytes was smaller and less abundant with no or a few granules. Both granulocytes and semigranulocytes had greater phagocytotic activity and a higher lysosomal content than hyalinocytes. The results declared that granulocytes and semigranulocytes were the main haemocytes involved in the cellular defence mechanism in S. constricta.

Establishing a density-based method to separate proliferating and senescent cells from bone marrow stromal cells.

To assist in the study of cellular aging, we established a new method of enriching physiologically aged bone marrow stromal cells (BMSCs) in animals of any age using a Percoll density gradient centrifugation technique. BMSCs from mice 2 months of age were isolated, and their cellular age determined (over 80% Scal-1+ CD29+ CD11b- CD45- CD105- and able to differentiate into osteoblasts, adipocytes, and chondrocytes). As proof –of principle, cells were aged in vitro and confirmed by low bromodeoxyuridine (BrdU) incorporation and senescence-associated β-galactosidase (SA-β-gal) staining. Proliferating cells were enriched in high-density gradient layers, and senescent cells were enriched in low-density gradient layers. We confirmed that over 80% of cells from the low-density gradient layer were senescent with SA-β-gal staining and telomere dysfunction-induced foci (TIF) assay. This density-based method, which can separate proliferating and senescent BMSCs, could be used to study mechanisms of physiologic cell aging and may have implications for the use of BMSCs in clinical transplant applications.

Isolation and purification of immune cells from the liver

Isolating and purifying liver immune cells are crucial for observing the changes in intrahepatic immune responses during the development of liver diseases and exploring the potential immunological mechanisms. Therefore, the aim of this study was to provide an optimal protocol for isolating immune cells with a high yield and less damage. We compared mechanical dissection and collagenase digestion, and the results were represented by the proportion of lymphocytes, Kupffer cells and neutrophils. The apoptosis rates of liver immune cells resulted by different isolation protocols were compared by Annexin V-staining using flow cytometric analysis. Our data indicated that the enzymatic digestion in vitro was more efficient than the mechanical dissection in vitro with a suitable collagenase IV concentration of 0.01%, and the purification of liver immune cells by a one-step density gradient centrifugation in 33% Percoll had the definite advantage of a higher proportion of the target cells. We also provided evidence that enzymatic digestion in vitro method was superior to collagenase digestion in situ for liver T lymphocytes, NK cells and NKT cells isolation and purification. This protocol was also validated in human liver samples. In conclusion, we developed an optimal protocol for isolating and purifying immune cells from mouse and human liver samples in vitro by 0.01% collagenase IV and 33% Percoll density gradient centrifugation with the advantages of higher cell yields and viability. This method provides a basis for further studying liver immune cells and liver immunity with a wide range of applications.

Purification and production of Plasmodium falciparum zygotes from in vitro culture using magnetic column and Percoll density gradient

BackgroundPlasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates.MethodsPlasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28.ResultsAfter stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28.ConclusionThe combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium.

Chloroplast Implication in the Tolerance to Salinity of the Halophyte Cakile maritima

In this study, the response of the chloroplastic antioxidant system of the halophyte Cakile maritima Scop. and its tolerance to NaCl stress have been studied using purified chloroplasts. Seedlings were grown in different salt concentrations (0, 100, 200 and 400 mmol/L NaCl) and plants were harvested after 40 days. Isolated chloroplasts were purified by centrifugation in density-gradients of Percoll. The evaluation of the oxidative stress was analysed measuring lipid peroxidation, carbonyl protein, $${\text{O}}_{2}^{ - }$$ and H2O2 contents and the antioxidant status by measurement of the activities of superoxide dismutase, catalase, peroxidase and enzymes of the ascorbate–glutathione cycle as well as the antioxidants ascorbate and glutathione, in the purified chloroplasts. Results revealed that the best growth response of C. maritima under moderate salt stress was associated with a low oxidative stress, the highest activities of SOD, POD and APX and the highest glutathione content. The interaction between the water status of the plant and mineral nutrition seems to be strongly involved in the plant performance under salinity.

STAT3 Localizes in Mitochondria-Associated ER Membranes Instead of in Mitochondria.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor (TF) that regulates a variety of biological processes, including a key role in mediating mitochondrial metabolism. It has been shown that STAT3 performs this function by translocating in minute amounts into mitochondria and interacting with mitochondrial proteins and genome. However, whether STAT3 localizes in mitochondria is still up for debate. To decipher the role of mitochondrial STAT3 requires a detailed understanding of its cellular localization. Using Percoll density gradient centrifugation, we surprisingly found that STAT3 is not located in the mitochondrial fraction, but instead, in the mitochondria-associated endoplasmic reticulum membrane (MAM) fraction. This was confirmed by sub-diffraction image analysis of labeled mitochondria in embryonic astrocytes. Also, we find that other TFs that have been previously found to localize in mitochondria are also found instead in the MAM fraction. Our results suggest that STAT3 and other transcriptional factors are, contrary to prior studies, consolidated specifically at MAMs, and further efforts to understand mitochondrial STAT3 function must take into consideration this localization, as the associated functional consequences offer a different interpretation to the questions of STAT3 trafficking and signaling in the mitochondria.

Low birth weight influences the postnatal abundance and characteristics of satellite cell subpopulations in pigs

Low birth weight (LBW) can cause lifelong impairments in muscle development and growth. Satellite cells (SC) and their progeny are crucial contributors to myogenic processes. This study provides new data on LBW in piglets combining insights on energy metabolism, muscle capillarization and differences in SC presence and function. To this aim, muscle tissues as well as isolated myogenic cells of 4-day-old German Landrace piglets were analyzed. For the first time two heterogeneous SC subpopulations, which contribute differently to muscle development, were isolated from LBW pigs by Percoll density gradient centrifugation. The muscles of LBW piglets showed a reduced DNA, RNA, and protein content as well as lower activity of the muscle specific enzymes CK, ICDH, and LDH compared to their normal birth weight siblings. We assume that deficits in energy metabolism and capillarization are associated with reduced bioavailability of SC, possibly leading to early exhaustion of the SC reserve cell pool and the cells’ premature differentiation.

Proteomic profiling of hepatic stellate cells in alcohol liver fibrosis reveals proteins involved in collagen production.

Hepatic stellate cell (HSC) activation has central functions in alcohol-induced liver fibrosis. Proteins of HSCs in alcoholic liver fibrosis (ALF) are still not completely understood. Here, we performed a proteomic study to discover proteins related to ALF using HSCs isolated from a rat model. Sprague-Dawley rats were fed with ethanol for 2 or 6 weeks. Liver histology was assessed using Sirius red and Oil red O staining. HSCs were enriched by using Percoll density gradient centrifugation, and analyzed using flow cytometry. Proteins extracted from HSCs were separated using two-dimensional electrophoresis (2DE). Differentially expressed proteins were identified using liquid chromatography-mass spectrometry (LC-MS). The characteristics of the differentially expressed proteins were analyzed using the UniProtKB database and STRING software. The mRNA levels of two differentially expressed proteins were analyzed using real-time RT-PCR, of which NADH dehydrogenase (ubiquinone) flavoprotein 2, mitochondrial (Ndufv2) was further investigated using Western blot (WB) and immunohistochemical analysis in the ALF model and human liver tissues. The relationship between Ndufv2 and alcohol stimulation was evaluated using WB. Next, Ndufv2 was knocked-down by shRNA in the HSC-T6 cell line. Three genes (encoding collagen, metalloproteinase inhibitor 1 [TIMP-1], and α-smooth muscle actin [a-SMA]) related to HSC activation were detected. An ALF model was successfully established, with a liver fibrosis score of 1-2 (S1-2), and some big fat vacuoles development. Twenty-one non-abundant proteins with more than a 2-fold difference were identified using mass spectrometry, including 7 upregulated and 14 downregulated proteins. These differential proteins are a response to antigen presentation, mitochondrial metabolism, ethanol, and collagen degradation. Among them, two upregulated proteins (Ndufv2 and ATP synthase subunit alpha, mitochondrial [ATP5a1]) were involved in mitochondrial metabolism in ALF, and showed concurrent changes in mRNA and protein levels. Ndufv2 was upregulated in HSCs, as shown by WB, in non-parenchymal cells (NPCs) in the rat model and human liver tissues, and detected using immunohistochemistry. Ndufv2 was also upregulated after alcohol stimulation. Following Ndufv2 knockdown, collagen, TIMP-1, and α-SMA were downregulated compared with that in the controls. A proteomic study was performed to discover proteins related to ALF in HSCs isolated from a rat model. Twenty-one differentially expressed proteins were identified, including proteins involved in mitochondrial metabolism and antigen presentation. Ndufv2, an upregulated protein in ALF, might be involved in ALF through regulating the production of fibrosis factors.

Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture

The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9<sup>th</sup>, 30<sup>th</sup> and 65<sup>th</sup> day) for the development of long-term culture method. The testes from 7–15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system.

A High Throughput Isolation Method for Phosphate-Accumulating Organisms

Hyperphosphatemia is a secondary issue associated with chronic kidney disorder. Use of phosphate binders and dialysis are the treatments for hyperphosphatemia, albeit with harmful side effects and high cost, respectively. A safer and healthier approach is attempted to administer phosphate-accumulating organisms (PAOs) from probiotics to prevent hyperphosphatemia. However, screening and isolation of PAOs are limited by inefficient enrichment of relevant metabolism and contamination. Therefore, we devised a novel strategy to isolate elite PAOs from Lactobacillus casei JCM 1134 and Bifidobacterium adolescentis JCM 1275 (previously reported PAOs). PAOs were first enriched for phosphate uptake and incubated in low-pH phosphate-free media to dormant non-PAOs, and then purified using Percoll density gradient centrifugation. Subsequently, elite PAOs were isolated from centrifuged pellet on a toluidine blue O-supplemented agar-based media. Using this technique, elite PAOs could not only be isolated, but also semi-quantitatively scored for their phosphate accumulation capabilities. Additionally, these scores correlated well with their accumulated phosphate values. The elite PAOs isolated from L. casei and B. adolescentis showed 0.81 and 0.70 [mg-phosphate/mg-dry cell], respectively (23- and 4.34-fold increase, respectively). Thus, our method can be used to successfully isolate elite PAOs, which might be of use to prevent hyperphosphatemia at early stages.

Artificial Insemination using liquid sperm Filial Ongole Bull after sexed with different methods

The purpose of this study was to determine the success of artificial insemination (AI) using liquid sperm Filial Ongole Bulls after sexed by the percoll density gradient centrifugation (PDGC) and egg white sedimentation (EWS) sexed methods. The study materials were 135 cattle were inseminated using non sexed semen, PDGC and EWS sexed methods. Non-return rate (NRR) and the conception rate (CR) are observed. The research design used for the success of AI using chi square. The result of pregnancy examination at the age of 3 months found 21 head of pregnant cows from 45 cattle in AI with without sexed (control), 14 head of pregnant cattle from 45 cattle in the AI with EWS method, 18 head of pregnant cattle from 45 cattle in AI with PDGC method. The result showed that CR value using liquid sperm result of EWS, PDGC and without sexed were very significant (P <0.01). CR value is still higher sperm without sexed (46.67%) followed by PDGC (40%) and EWS (31.11%). The value of conception rate is lower than sexed using PDGC method. The success of AI showed better PDGC sexed method than EWS sexed method based on AI liquid sperm results with CR analysis.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ± 4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ± 0.25%) and lipofectamine 3000 (15.57 ± 0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.

Tingkat keberhasilan Inseminasi Buatan (IB) menggunakan semen beku hasil sexing pada Sapi Persilangan Ongole

The purpose of artificial insemination using sexing semen was to obtain the calf according to the expected sex. This study aimed to determine the success of artificial insemination in non-sexing and sexing of frozen semen. The materials used in this research were 77 non-sexing artificial insemination acceptor cows and 77 sexing artificial insemination acceptor cows with double dose treatment. The method used in this study was experimental field experiment with the research treatment: P0 = frozen non-sexing semen straw and P1 = frozen semen of spermatozoa Y by sexing method using Percoll Density Gradient Centrifugation (SGDP) produced by Balai Besar Inseminasi Buatan (BBIB) Singosari. The variables of this study were Non Return Rate (NRR), Service Per Conception (S/C) and Conception Rate (CR). The data obtained were analyzed descriptively. The results of the study in each non-sexing and sexing artificial insemination treatment were: the percentage of NRR 1 were 90.91% and 77.92%, followed by NRR 2 with 88.31% and 76.62%, the pregnancy percentage were 84.42% and 75.33%, and the CR values were 79.22% and 57.14%. The conclusion of this study was artificial insemination using frozen non-sexing semen had a higher success rate compared to artificial insemination using frozen sexing semen.

Differential proteomic profiles and characterizations between hyalinocytes and granulocytes in ivory shell Babylonia areolata

The haemocytes of the ivory shell, Babylonia areolata are classified by morphologic observation into the following types: hyalinocytes (H) and granulocytes (G). Haemocytes comprise diverse cell types with morphological and functional heterogene and play indispensable roles in immunological homeostasis of invertebrates. In the present study, two types of haemocytes were morphologically identified and separated as H and G by Percoll density gradient centrifugation. The differentially expressed proteins were investigated between H and G using mass spectrometry. The results showed that total quantitative proteins between H and G samples were 1644, the number of up-regulated proteins in G was 215, and the number of down-regulated proteins in G was 378. Among them, cathepsin, p38 MAPK, toll-interacting protein-like and beta-adrenergic receptor kinase 2-like were up-regulated in G; alpha-2-macroglobulin-like protein, C-type lectin, galectin-2-1, galectin-3, β-1,3-glucan-binding protein, ferritin, mega-hemocyanin, mucin-17-like, mucin-5AC-like and catalytic subunit of protein kinase A were down-regulated in G. The results showed that the most significantly enriched KEGG pathways were the pathways related to ribosome, phagosome, endocytosis, carbon metabolism, protein processing in endoplasmic reticulum and oxidative phosphorylation. For phagosome and endocytosis pathway, the number of down-regulation proteins in G was more than that of up-regulation proteins. For lysosome pathway, the number of up-regulation proteins in G was more than that of down-regulation proteins. These results suggested that two sub-population haemocytes perform the different immune functions in B. areolata.

Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol.

The oleaginous yeast Lipomyces starkeyi is an attractive organism for the industrial production of lipids; however, the amount of lipid produced by wild-type L. starkeyi is insufficient. The study aims to obtain L. starkeyi mutants that rapidly accumulate large amounts of triacylglycerol (TAG). Mutagenized yeast cells at the early stages of cultivation were subjected to Percoll density gradient centrifugation; cells with increased production of TAG were expected to be enriched in the resultant upper fraction because of their lower density. Among 120 candidates from the upper fractions, five mutants were isolated that accumulated higher amounts of TAG. Moreover, when omitting cells with mucoid colony morphology, 11 objective mutants from 11 candidates from the upper fraction were effectively (100%) isolated. Of total 16 mutants obtained, detailed characterization of five mutants was performed to reveal that five mutants achieved about 1.5-2.0 times TAG concentration (4.7-6.0g/L) as compared with the wild-type strain (3.6g/L) at day 5. Among these five mutants, strain E15 was the best for industrial use because only strain E15 showed significantly higher TAG concentration as well as significantly higher degree of lipid to glucose and biomass to glucose yields than the wild-type strain. Thus, Percoll density gradient centrifugation is an effective method to isolate mutant cells that rapidly accumulate large amounts of TAG. It is expected that by repeating this procedure as part of a yeast-breeding program, L. starkeyi mutants suitable for industrial lipid production can be easily and effectively obtained.

Density Based Characterization of Mechanical Cues on Cancer Cells Using Magnetic Levitation.

Extracellular matrix (ECM) stiffness is correlated to malignancy and invasiveness of cancer cells. Although the mechanism of change is unclear, mechanical signals from the ECM may affect physical properties of cells such as their density profile. The current methods, such as Percoll density-gradient centrifugation, are unable to detect minute density differences. A magnetic levitation device is developed (i.e., MagDense platform) where cells are levitated in a magnetic gradient allowing them to equilibrate to a levitation height that corresponds to their unique cellular density. In application of this system, MDA-MB-231 breast and A549 lung cancer cells are cultured and overall differences in cell density are observed in response to increasing collagen fiber density. Overall, density values are significantly more spread out for MDA-MB-231 cells extracted from the 1.44 mg mL-1 collagen gels compared to those from 0.72 mg mL-1 collagen, whereas no significant difference with A549 cell lines is observed. The MagDense platform can determine differences in cellular densities under various microenvironmental conditions. The imaging of cancer cells in a magnetic levitation device serves as a unique tool to observe changes in phenotypic properties of cancer cells as they relate to micromechanical cues encoded by the ECM.

The Motility and Ratio of X and Y Sperm Filial Ongole Cattle Using Different Sexed Semen Methods

Artificial Insemination (AI) using sexed semen produce sex of calf as expected. There are various methods of sexed semen has been found. The aim of this research is to determine the effect of sexed semen methods using egg white sedimentation and percoll density gradient centrifugation toward motility and ratio of X and Y sperm Filial Ongole Cattle. Semen was collected using Artificial Vagina (AV) from Filial Ongole Cattle, evaluated and then separated using egg white sedimentation and percoll density gradient centrifugation technique. Only fresh semen with a minimum of 70% individual motile sperm and 2+ mass motility used in this study. Andromed as a based extender was diluted using aquabidest with 1:4 ratio. The obtained data were analyzed with analysis of variance (ANOVA) and continued by Duncan test if there was significant or very significant different. The result showed that the sexing methods (non sexing sperm, upper and under fraction using percoll density gradient centrifugation, upper and under fraction using egg white sedimentation technique) had very significant effect (P<0.01) on motility (64.25±3.94%; 48.55±8.28%; 53±7.93%; 56.9±8.22%; 49.75±8.19% respectively) and significant effect (P<0.05) on the ratio of X and Y sperm. The ratio of X and Y using percoll density gradient centrifugation and egg white sedimentation methods for the upper fraction were 28.0±2.26%:72.0±2.26% and 77.5±1.26%:22.5±1.26%. The ratio of X and Y using percoll density gradient centrifugation and egg white sedimentation technique for the under fraction were 69.0±15.35%: 26.0±4.37% and 22.9±1.44%: 77.1±1.44%. The study concludes that the sexed with egg white sedimentation technique is better than percoll density gradient centrifugation on sperm motility and ratio of X and Y sperm.