Cell separation

We present a model of the mechanical and fluid forces associated with exudative retinal detachments where the retinal photoreceptor cells separate, typically from the underlying retinal pigment epithelium (RPE). By computing the total fluid volume flow arising from transretinal, vascular and RPE pump currents, we determine the conditions under which the subretinal fluid pressure exceeds the maximum yield stress holding

In this work, a brewer’s yeast strain was used to remove heavy metals from a synthetic effluent. The solid–liquid separation process was carried out using the flocculation ability of the strain. The yeast strain was able to sediment in the presence of Cu2+, Ni2+, Zn2+, Cd2+ and Cr3+, which evidences that the flocculation can be used as a cheap and natural separation process for an enlarged range of industrial effluents. For a biomass concentration higher than 0.5 g/l, more than 95% of the cells were settled after 5 min; this fact shows that the auto-aggregation of yeast biomass is a rapid and efficient separation process. Cells inactivated at 45 °C maintain the sedimentation characteristics, while cells inactivated at 80 °C lose partially (40%) the flocculation.The passage of metal-loaded effluent through a series of sequential batches allowed, after the second batch, the reduction of the Ni2+concentration in solution for values below the legal limit of discharge of wastewater in natural waters (2 mg/l); this procedure corresponds to a removal of 91%. A subsequent batch had a marginal effect on Ni2+ removal (96%).Together, the results obtained suggest that the use of brewing flocculent biomass looks a promising alternative in the bioremediation of metal-loaded industrial effluents since the removal of the heavy metals and cell separation are simultaneously achieved.

The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 c...

Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction.Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies.In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV = 2.58%) and reproducibility (CV = 2.57%) was shown.The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P > 0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11 ± 0.56% for ejaculates and 50.17 ± 0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-pr...