Magnetic field design for selecting and aligning immunomagnetic labeled cells

BACKGROUND: Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. RESULTS: A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. CONCLUSIONS: The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.     

Cell analysis system based on compact disk technology

BACKGROUND: A cell analysis system was developed to enumerate and differentiate magnetically aligned cells selected from whole blood. The cellular information extracted is similar to the readout of musical information from a compact disk (CD). Here we describe the optical design and data processing of the system. The performance of the system is demonstrated using fluorescent-labeled cells and beads. Materials and Methods System performance was demonstrated with 6-microm polystyrene beads labeled with magnetic nanoparticles and allophycocyanin (APC) and immunomagnetically aligned leukocytes, fluorescently labeled with Oxazine750 and CD4-APC, CD8-Cy5.5, and CD14-APC/Cy7 in whole blood. RESULTS: The sensitivity of the system was demonstrated using APC-labeled beads. With this system, beads containing 333 APC molecules could easily be resolved from the background. This level of sensitivity was not achievable with a commercial flow cytometer. A maximum of 20,000 immunomagnetically labeled cells could be aligned and analyzed in between 0.6 m of Ni lines, distributed over a surface area of 18 mm(2) and extracted from a blood volume that depended on the height of the chamber. The utility of the system was demonstrated by performing a three-color CD4-CD8-CD14 assay. CONCLUSIONS: We built a cell analysis system based on immunomagnetic cell selection and alignment and analysis of fluorescent signals employing CD-technology that is as good or better than current commercial analyzers. The cell analysis can be performed in whole blood or any other type of cell suspension without extensive sample preparation.     

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.     

Ease of calving, blood chemistry, insulin and bovine growth hormone of newborn calves derived from embryos produced in vitro in culture systems with serum and co-culture or with PVA

Blood chemistry (pH, pCO2, pO2, glucose, lactate) as well as plasma insulin and growth hormone of calves derived from embryos produced under 2 different in vitro culture systems (modified SOFaa with 20% serum and co-culture with bovine oviduct epithelial cells [IVP serum, n=8] or with 3 mg/mL PVA [IVPdefined, n=6]) were compared with those of calves derived from AI (n=5). Calvings were classified according to the ease (unassisted, light traction, heavy traction). Blood samples were taken from the jugular vein of calves at 5, 15, 30 and 60 min, and at 2, 3, 6, 12, 18 and 24 h after delivery, then daily for 6 d. At the second day of life after 4 feedings and a 4-h fasting period, a glucose tolerance test was performed to evaluate glucose metabolism and insulin secretion. Calves in the IVP serum group had higher birth weights than AI calves (LS mean +/- SEM, IVP serum: 45.2 +/- 1.4 kg vs AI: 40.4 +/- 1.7 kg; P < 0.05), while the birth weights of calves in the IVP defined group were in between (IVPdefined: 41.9 +/- 1.6 kg). More IVP serum calves (75%) needed assistance than IVP defined (33%) or AI (40%) calves. The effect of ease of calving vs the effect of embryo culture was compared in relation to blood parameters at birth. There was an effect of ease of calving but not of embryo culture conditions on blood pH, lactate and PCO2. Calves requiring heavy traction had lower pH during the first 3 h after calving, a higher lactate during the first 60 min after calving and a higher pCO2 the first 2 h after calving than calves born unassisted. Calves requiring heavy traction also had lower pH the first 2 h and higher lactate the first 3 h after calving than calves born by light traction. IVP defined calves had lower lactate than IVP serum calves the first 60 min after calving. At 6 h after delivery, all blood parameters had stabilized. There was no effect of either embryo culture or ease of calving on basal insulin and growth hormone level, or the ability of the calves to handle glucose postnatally and during a glucose tolerance test.     

Body dimensions and birth and organ weights of calves derived from in vitro produced embryos cultured with or without serum and oviduct epithelium cells

Body dimensions, birth and organ weights of calves derived from embryos produced in 2 in vitro culture systems (modified SOFaa with 20% cattle serum and co-cultured with oviduct-epithelium cells [IVPserum, n=8], and modified SOFaa with 3 mg/mL PVA [IVPdefined, n=6]) were compared with calves originating from artificial insemination (AI, n=85). Three additional IVP calves were included which had been vitrified as mature oocytes by the open pulled straw (OPS) method, warmed, fertilized and cultured to the blastocyst stage in modified SOFaa with 5% cattle serum, then again OPS-vitrified and warmed prior to transfer (IVPops, n=3). At birth, gestation length and birth weights were registered for all calves. At 1 wk of age all 17 IVP and 7 of the AI calves were killed, and their body dimensions and organ weights recorded. Birth weight was higher for the IVPserum and IVPops calves than for AI control calves (kg +/- SEM: IVPserum 46.9+/-1.8, IVPops 50.6+/-2.4, AI 41.8+/-0.8; P < 0.002). There was no difference between IVP and AI calves regarding gestation length and no effect of culture conditions on body dimensions or organ weights, except for longer hind legs in IVPdefined calves compared with AI calves (cm +/- SEM: IVPdefined 93+/-2, AI 87+/-2; P < 0.04). The IVPops calves had an increased liver weight compared with AI and the other IVP calves (g +/- SEM: IVPops 1.457+/-59; AI 1,117+/-37; IVPserum 1,159+/-34, IVPdefined 1,073+/-39; P < 0.0003). It is concluded that in vitro culture of bovine embryos in the presence of serum and oviduct epithelium cells increased birth weight but not organ weight and body dimension in 1-wk-old calves. However, vitrification of the ova as oocyte and again as blastocysts increased birth weight and liver size. This possible effect of cryopreservation of oocytes on subsequent fetal development awaits further investigation.     

Molecular systematics and biogeography of the fanged frogs of Southeast Asia

Our analysis of parts of the mitochondrial ribosomal 12S and 16S genes from 39 populations of Southeast Asian ranid frogs confirms that the fanged frogs are a monophyletic clade. This group, properly called Limnonectes, appears to have arisen in the early Tertiary at a time when free faunal exchange was possible among Southeast Asia, Borneo, Sumatra, Java, and, probably, Sulawesi. Four species groups are tentatively identified within the clade. Part of group 1 includes species related to L. kuhlii that occur in Borneo. Another part of group 1 includes species from Malay Peninsula and Thailand that are related to L. pileata. Species group 2, L. leporina, occurs only in Borneo. Species group 3 is restricted to species distributed in Sulawesi and the Philippines. Species group 4 includes L. blythii and relatives. There is a lack of compatibility between phylogenetic hypotheses generated from molecular and morphological data sets. These differences are related, in large part, to whether some species of Limnonectes have secondarily lost fangs or whether lack of fangs represents the primitive condition.     

Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination

Availability of embryos of high quality is required to obtain satisfactory embryonic developmental rates and normal calves following transfer of in vitro-produced (IVP) bovine embryos. One relevant quality parameter is the frequency of chromosome aberrations, which can be evaluated using multicolor fluorescent in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes in cattle. In this study, interphase nuclei (n = 3805) were analyzed from 426 bovine IVP embryos. We found that 73%, 72%, 81%, and 58% of the embryos from Days 2, 3, 4, and 5 post-insemination (pi), respectively, displayed a normal diploid chromosome number in all cells. When looking at the types of chromosome aberrations, the percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of polyploidy (i.e., all nuclei in an embryo were analyzed and were polyploid) were 5%, 13%, 3%, and 0%, respectively. In conclusion, numerical chromosome aberrations were detected as early as Day 2 pi. The development of polyploid embryos is slow and is apparently arrested during the third cell cycle, whereas the mixoploid embryos seem to continue development.     

Biomolecular interactions measured by atomic force microscopy

Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites. In this review we discuss the important contributions that have led to the development of this field. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible measurements by AFM. This increased reproducibility will pave the way for a better understanding of molecular interactions in cell biology.     

Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscope.

Photolyase DNA interactions and the annealing of restriction fragment ends are directly visualized with the atomic force microscope (AFM). To be able to interact with proteins, DNA must be loosely bound to the surface. When MgCl2 is used to immobilize DNA to mica, DNA is attached to the surface at distinct sites. The pieces of DNA in between are free to move over the surface and are available for protein interaction. After implementation of a number of instrumental improvements, the molecules can be visualized routinely, under physiological conditions and with molecular resolution. Images are acquired reproducibly without visible damage for at least 30 min, at a scan rate of 2 x 2 microm2/min and a root mean square noise of less than 0.2 nm. Nonspecific photolyase DNA complexes were visualized, showing association, dissociation, and movement of photolyase over the DNA. The latter result suggests a sliding mechanism by which photolyase can scan DNA for damaged sites. The experiments illustrate the potential that AFM presents for modern molecular biology.