Percoll Density Centrifugation Research Articles

Quality and Proportion of Spermatozoa in the Percoll Density Gradient Centrifugation Sexing Method at Different Gradients using AndroMed® Diluent

The utilization of sexing technology is an effort made to improve the efficiency of livestock farming, which was created to predict the sex of the calves born so that it can be adjusted to the objectives of the farm. This study aims to analyze the quality and proportion of sexed spermatozoa using the percoll density centrifugation method at different gradients with AndroMed® diluent. The research material used was fresh semen from Belgian Blue Bull aged six years with a body weight of 600 kg and fresh semen quality motility ≥70%. This research is a laboratory experiment with two treatments and ten replicates. The treatments in the study were T0: Ten Gradient density percoll + AndroMed® and T1: Five Gradient density percoll + AndroMed®. The research analysis used a dependent t-test. The statistical analysis showed a significant difference (P<0.05) in treating ten gradients with five gradients in each layer regarding individual motility, viability, abnormality, and total motile spermatozoa. At the same time, in concentration, there was no difference (P>0.05) in the treatment of 10 gradients with five gradients. The average results on individual motility, abnormality, concentration, and total motile spermatozoa showed treatment of 10 gradients better than treatment of 5 gradients in each layer. At the same time, the variable viability showed that the gradient treatment is better than the five-gradient treatment. Proportion of spermatozoa in the Upper Layer T0 X (23.4%), Y (76.8%) and T1 X (16.9%), Y (83.1%). The proportion of Spermatozoa in the Bottom Layer was T0 X (84.1%), Y (15.9%), and T1 X (83%), Y (17%), respectively. In conclusion, sexing spermatozoa with the Percoll 10 and 5 Gradient Density Centrifugation method can separate X and Y spermatozoa.

An optimized method for the induction and purification of mouse bone marrow dendritic cells

Dendritic cells (DCs) play an essential role in the initiation of adaptive immune responses, but they are rare in all organs. The traditional methods used to increase the yield and purity of DCs are the early removal of granulocyte culture medium and the isolation of high-purity DCs by magnetic-activated cell sorting (MACS). This study provides a more rapid and economical optimization method to obtain more high-purity DCs. (i) We harvested 18% more bone marrow (BM) cells by using forceps to crack the epiphysis instead of cutting it with scissors during BM cell extraction. (ii) When the cells in the culture medium that is discarded on day 3 in the traditional method were centrifuged and then added back to the petri dish, the DC yield on day 5 increased by 61%. (iii) On the third day, the addition of fresh medium and the retention of the original medium rather than discarding it increased the number of DCs harvested on the fifth day by 137%. (i-iii) The improved method cost an average of 74% less than the conventional method and yielded the same number and function of cells. (iv) The initial number of BM cells was increased by 15% in 4-week-old mice compared with 8-week-old mice. (v) The Percoll density centrifugation (PDS) method was used to purify DCs on day 6 after induction, and the purity of the DCs was greater than 90%, which showed no significant difference from the MACS method. However, the yield of the PDS method increased by 21%. In addition, the PDS method has a lower cost, with an average purification cost of 4 CNY ($0.58) compared with 648 CNY ($93.25) for MACS, reducing the cost by 99%. Therefore, high-purity and high-yield DCs can be rapidly obtained through a five-step improvement in the process of BM cell extraction, induction and purification.

Optimization and enrichment of induced cardiomyocytes derived from mouse fibroblasts by reprogramming with cardiac transcription factors.

Ischemic heart disease within developed countries has been associated with high rates of morbidity and mortality. Cell‑based cardiac repair is an emerging therapy for the treatment of cardiac diseases; however, a limited source of the optimal type of donor cell, such as an autologous cardiomyocyte, restricts clinical application. The novel therapeutic use of induced pluripotent stem cells (iPSCs) may serve as a unique and unlimited source of cardiomyocytes; however, iPSC contamination has been associated with teratoma formation following transplantation. The present study investigated whether cardiomyocytes from mouse fibroblasts may be reprogrammed invitro with four cardiac transcription factors, including GATA binding protein4, myocyte‑specific enhancer factor2C, T‑box transcription factor5, and heart‑ and neural crest derivatives‑expressed protein2 (GMTH). Cardiac‑specific markers, including α‑myosin heavy chain (α‑MHC), β‑MHC, atrial natriuretic factor, NK2 homeobox5 and cardiac troponin T were observed within mouse fibroblasts reprogrammed with GMTH, which was reported to be more effective than GMT. In addition, Percoll density centrifugation enriched a population of ~72.4±5.5% α‑MHC+ induced cardiomyocytes, which retained the expression profile of cardiomyocyte markers and were similar to natural neonatal cardiomyocytes in well‑defined sarcomeric structures. The findings of the present study provided a potential solution to myocardial repair via a cell therapy applying tissue engineering with minimized risks of immune rejection and tumor formation.

Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function.

Retroviral (RV) expression of genes of interest (GOIs) is an invaluable tool and has formed the foundation of cellular engineering for adoptive cell therapy in cancer and other diseases. However, monitoring of transduced T cells long term (weeks to months) in vivo remains challenging because of the low frequency and often poor durability of transduced T cells over time when transferred without enrichment. Traditional methods often require additional overnight in vitro culture after transduction. Moreover, in vitro-generated effector CD8+ T cells enriched by sorting often have reduced viability, making it difficult to monitor the fate of transferred cells in vivo. Here, we describe an optimized mouse CD8+ T-cell RV transduction protocol that uses simple and rapid Percoll density centrifugation to enrich RV-susceptible activated CD8+ T cells. Percoll density centrifugation is simple, can be done on the day of transduction, requires minimal time, has low reagent costs and improves cell recovery (up to 60%), as well as the frequency of RV-transduced cells (∼sixfold over several weeks in vivo as compared with traditional methods). We have used this protocol to assess the long-term stability of CD8+ T cells after RV transduction by comparing the durability of T cells transduced with retroviruses expressing each of six commonly used RV reporter genes. Thus, we provide an optimized enrichment and transduction approach that allows long-term in vivo assessment of RV-transduced T cells. The overall procedure from T-cell isolation to RV transduction takes 2 d, and enrichment of activated T cells can be done in 1 h.

Abstract 472: A digital sorting-based detection and isolation assay for single disseminated cancer cells from lymph nodes of melanoma patients

Abstract We recently showed that sentinel lymph node (SLN) disaggregation followed by immunocytology enables precise quantification of disseminated cancer cells (DCC). We demonstrated that this approach has a 20-fold higher sensitivity to detect melanoma DCCs than routine histopathology and that the DCC density (DCCD, defined as the number of DCCs per million lymph node cells) together with ulceration state and tumor thickness enables individual risk prediction superior to current AJCC guidelines (Ulmer et al., PLoS Medicine, 2014). Here, we present the adaptation of this method to a semi-automated workflow, including automated single cell detection and isolation by DEPArrayTM technology for subsequent molecular analysis of DCCs from SLN of melanoma patients. The developed workflow includes a mechanical disaggregation of lymph node tissue and collection of the mononuclear cells after Percoll density centrifugation. Several tumor cell enrichment methods were tested (CellSearch® and size-based enrichment technologies), however, failed to process cell suspensions generated from lymph nodes due to clumping of cells during the enrichment procedure. Tumor cell enrichment using MACS depletion of CD45+ cells delivered approx. 50% of spiked-in cell line cells with a mean depletion rate of lymphocytes and erythrocytes > 98%. To prevent limitations in DCC detection caused by phenotypic heterogeneity of tumor cells, we established a double staining against two melanoma-associated markers gp100 and MCSP. Individual melanoma cells are then detected and isolated by DEPArrayTM technology enabling single cell whole genome amplification (Ampli1TM) for subsequent molecular analysis as assessment of copy number alterations (e.g. by arrayCGH) or sequence analysis for melanoma specific point mutations (e.g. BRAF or NRAS). We successfully applied the workflow to first SLN samples from melanoma patients. The established workflow enables a standardized detection of single DCCs from lymph nodes of melanoma patients applicable in clinical diagnostic labs. Automated single cell isolation and subsequent molecular analysis of DCCs is feasible within short time after SLN biopsy. In the future, this approach could help to select individualized therapies for melanoma patients. Citation Format: Sebastian Scheitler, Kathrin Weidele, Barbara Alberter, Christian Werno, Christoph A. Klein, Bernhard Polzer. A digital sorting-based detection and isolation assay for single disseminated cancer cells from lymph nodes of melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 472.

Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods

ABSTRACTStudies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

Release of Cell-Free Ice Nucleators from Three Recombinant Ice+Zymomonas mobilis Strains

Background/Aims: This work is a study of the ability of three recombinant Zymomonas mobilis strains to release ice nucleators into their growth medium. Methods: The recombinant ice⁺Z. mobilis cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. Results: Cell-free ice nucleators were obtained from all the recombinant ice⁺Z. mobilis cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 μm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. Conclusion: These findings indicate that the recombinant ice⁺Z. mobilis cells could be valuable for future use as a source of active cell-free ice nucleation protein.

Human peripheral neutrophils express functional IL-21 receptors.

Neutrophils play key roles in the inflammatory response. The IL-21 cytokine and receptor system is known to be involved in various inflammatory diseases. However, the direct action of IL-21 on neutrophils has not been reported. Here, we show that human neutrophils in peripheral blood express functional IL-21 receptors (IL-21Rs). Expression of the IL-21Rα chain (IL-21Rα) was reduced following various treatments to remove red blood cells, including hypotonic shock, ammonium chloride-mediated lysis, and Percoll density centrifugation. Thus, we utilized whole blood flow cytometric assays to investigate the neutrophil responses to IL-21. IL-21 upregulated the surface expression of CD11b and CD16 on neutrophils and augmented the neutrophils' phagocytic ability. Our data indicated that IL-21 has the potential to enhance the neutrophil functions.

An alternative protocol for Plasmodium falciparum culture synchronization and a new method for synchrony confirmation

BackgroundAlthough the Percoll/sorbitol synchronization method is widely accepted, its use for achieving tight synchronizations is cumbersome. In addition, subsequent conclusions on the synchrony status are often based on visual inspection of parasites and few reports provide an unbiased estimate confirming the degree of synchrony. This report presents a simpler synchronization procedure and offers an objective method to validate parasite synchrony.MethodsParasite synchronization was performed by culturing late-stage schizont parasites for a defined period of time, subjecting them to Percoll density centrifugations, and collecting the newly formed rings. Repeating the process several times on the un-egressed schizonts maximizes the recovery of several synchronized ring-stage parasite populations. The culture synchrony for each population was verified by allowing the synchronized rings to mature to late-stage schizonts and collecting ring-stage sample aliquots at three-hour intervals for nine hours. The aliquots were then measured, using the SyBr Green I assay, to determine when the ring-stage parasitaemia stops increasing.ResultsQuantitative measurements of ring-stage parasites showed that under the conditions described, a four to six-hour synchrony period is obtained.ConclusionBy taking advantage of Plasmodium’s periodic lifecycle in erythrocytes, it is shown that Percoll density centrifugation alone is sufficient to tightly synchronize cultures with minimal parasite loss. In addition, the degree of culture synchrony is validated using the SyBr Green I assay.

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

Factors influencing the density of aerobic granular sludge

In the present study, the factors influencing density of granular sludge particles were evaluated. Granules consist of microbes, precipitates and of extracellular polymeric substance. The volume fractions of the bacterial layers were experimentally estimated by fluorescent in situ hybridisation staining. The volume fraction occupied by precipitates was determined by computed tomography scanning. PHREEQC was used to estimate potential formation of precipitates to determine a density of the inorganic fraction. Densities of bacteria were investigated by Percoll density centrifugation. The volume fractions were then coupled with the corresponding densities and the total density of a granule was calculated. The sensitivity of the density of the entire granule on the corresponding settling velocity was evaluated by changing the volume fractions of precipitates or bacteria in a settling model. Results from granules originating from a Nereda reactor for simultaneous phosphate COD and nitrogen removal revealed that phosphate-accumulating organisms (PAOs) had a higher density than glycogen-accumulating organisms leading to significantly higher settling velocities for PAO-dominated granules explaining earlier observations of the segregation of the granular sludge bed inside reactors. The model showed that a small increase in the volume fraction of precipitates (1-5%) strongly increased the granular density and thereby the settling velocity. For nitritation-anammox granular sludge, mainly granular diameter and not density differences are causing a segregation of the biomass in the bed.

Analysis of time-course gene expression profiles of sinusoidal endothelial cells during liver regeneration in rats

Liver regeneration (LR) after partial hepatectomy (PH) requires the coordinate contribution of different cell types. Liver sinusoidal endothelial cells (LSECs), representing the largest population of nonparenchymal cells, are proven to be crucial in LR. However, the details about their implications in regeneration are not still clear. In this study, percoll density centrifugation and immunomagentic bead methods were used to isolate LSECs with high purity and yield; global transcriptional profiles of LSECs during the regeneration were investigated by microarray. 1,629 genes were identified to be LR-related. Among them, there were 833 known genes whose expression patterns were clustered into eight classes. Gene function enrichment analysis showed that genes involved in the major LSEC functions, i.e., coagulation, phagocytosis, and transport, were highly enriched in cluster characterized by rapid induction and gradual return, suggesting the quick reestablishment of LSEC function after PH. Genes in immunity/inflammation and defense response were enriched in clusters exhibiting transient downregulation and quick recovery, possibly being associated with suppression of immunity/inflammation pathway in LSECs at early phase. Genes in glycogen synthesis and glycolysis were enriched in the clusters marked by "significant increase and gradual return" and "slight increase and then downregulation", implying an enhanced carbohydrate metabolism at early phase; detoxification-related genes were markedly distributed in the cluster with feature of rapid increase and then reduction, which was helpful in eliminating waste substance. Taken together, the measurement of gene expression profiling of LSECs and expression pattern analysis of functionally categorized genes gave insight into the mechanism of action of this cell on LR.

Differential gene expression and functional analysis of pit cells from regenerating rat liver

Hepatic pit cells are a population of large granular lymphocytes that substantially contribute to hepatic immunity. Studies have proven that pit cells have a role in liver regeneration, but the details of the relationship between pit cells and liver regeneration is not clear at present. We subjected rats to a two-third hepatectomy; pit cells with high purity were obtained with Percoll density centrifugation and immunomagnetic bead methods, and the changes in mRNA levels in pit cells from the regenerating liver were monitored up to 168 h using a Rat Genome 230 2.0 Array composed of 25,020 distinct rat liver cDNA clones. Of the 25,020 genes analyzed, 612 known and 358 unknown genes were identified to be associated with liver regeneration. The 612 known genes are classified into up-regulation and down-regulation patterns based on the expression levels; they primarily participate in at least 23 biological activities based on gene ontology analysis. Together with gene function enrichment analysis, cytokines and a growth factor-mediated pathway in pit cells were activated at an early phase of liver regeneration; pit cell proliferation occurred from 24-72 h after liver hepatectomy; the machinery of pit cell differentiation commenced early and came into play late; an immune/inflammatory response was enhanced late. Expression pattern analysis of functionally classified genes in pit cells can give insights into the relationship between pit cells and liver regeneration.

A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94–100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9–97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. It has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5%±1.9% (n=11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8±2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.

Cultured trout gill epithelia enriched in pavement cells or in mitochondria-rich cells provides insights into Na+ and Ca2+ transport

The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kOmega cm(2)) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na(+) fluxes and application of the Ussing flux ratio criterion demonstrated active Na(+) uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca(2+) uptake and Na(+)/K(+)-ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.

Effect of tungsten and molybdenum on growth of a syntrophic coculture of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H2/CO2 required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H2 as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.

Purification of human megakaryocytes using a glycoprotein Ib dependent agglutination technique

In order to study normal and abnormal human megakaryocytopoiesis we modified a glycoprotein Ib dependent megakaryocyte purification technique originally described in guinea pigs. After Percoll density centrifugation, 2 ml bone marrow aspirates were subjected to 3 steps of bovine plasma induced cell clumping, which should selectively agglutinate glycoprotein Ib positive cells. In 13 experiments the resulting samples contained 28-102 x 10(3) megakaryocytes (median 70) with a median purity of 79%. Megakaryocyte recovery ranged from 30-86% (median 51%). The DNA profile showed median values of 10% for 8 N, 44% for 16 N and 43% for 32 N megakaryocytes. The described method is relatively simple, inexpensive and gives results comparable with more elaborate techniques.

Anion Channels, Including ClC-3, Are Required for Normal Neutrophil Oxidative Function, Phagocytosis, and Transendothelial Migration

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.