Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry.

BACKGROUND: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. METHODS: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. RESULTS: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde/saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. CONCLUSION: Paraformaldehyde/saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations.     

Activity-dependent plasticity of synaptic efficacy at inhibitory junctions

Despite a considerable amount of investigation on long-term potentiation, the question of whether this process occurs at inhibitory synapses has remained controversial until studies of these junctions have been achieved in the Mauthner cell of Teleosts. In this preparation, inhibitory long-term potentiation similar to that occurring at hippocampal excitatory synapses has been demonstrated.     

Cell blocks in cytopathology: a review of preparative methods,utility in diagnosis and role in ancillary studies

The cell block (CB) is a routine procedure in cytopathology that has gained importance because of its pivotal role in diagnosis and ancillary studies. There is no precise review in the published literature that deals with the various methods of preparation of CB, its utility in diagnosis, immunocytochemistry (ICC) or molecular testing, and its drawbacks. An extensive literature search on CB in cytology using internet search engines was performed for this review employing the following keywords: cell block, cytoblock, cytology, cytopathology, methods, preparation, fixatives, diagnostic yield, ancillary and molecular studies. Ever since its introduction more than a century ago, the CB technique has undergone numerous modifications to improve the quality of the procedure; however, the overall principle remains the same in each method. CBs can be prepared from virtually all varieties of cytological samples. In today's era of personalized medicine, cytological specimens, including CBs, augment the utility of cytological samples in analysing the molecular alterations as effectively as surgical biopsies or resection specimens. With the availability of molecular targeted therapy for many cancers, a large number of recent studies have used cytological material or CBs for molecular characterization. The various techniques of CB preparation with different fixatives, their advantages and limitations, and issues of diagnostic yield are discussed in this review.     

The art and artifact of GDF9 activity: cumulus expansion and the cumulus expansion-enabling factor

The process of cumulus cell expansion is critical for normal fertility. Oocyte-produced growth and differentiation factor 9 (GDF9) has been thought to play a leading role in this process. Recent studies both support and refute this hypothesis. Central to understanding the physiology of GDF9 is the use of recombinant ligand in in vitro assays. There are several laboratories that currently produce recombinant GDF9 preparations that appear to show variable effects on granulosa cell gene expression and cumulus cell expansion. Several of these studies are reviewed here. Standardization in preparation for recombinant GDF9, as well as a more biochemical analysis of the oocyte-secreted forms of GDF9, may help to resolve the conflicts currently seen in the literature.     

Culture of intramural cardiac ganglia of the newborn guinea-pig

The ultrastructure of cultured intrinsic neurones and SIF (small intensely fluorescent) cells dissociated from the atria and interatrial septum of newborn guinea-pig heart has been studied for the first time and compared with these cells in situ. Mononucleate and binucleate neuronal somata and their processes were observed in the culture preparation; their ultrastructure was similar to that of neurones in intracardiac ganglia observed in situ. The number of neurites associated with neuronal cell bodies increased after the first week in culture. A subpopulation of intracardiac neurones showed abnormalities in culture, comparable to the changes previously described in neurones of the monkey heart after unilateral vagotomy in situ. Small granule-containing cells were observed in culture, corresponding to those described in the heart in situ. One type of large process in the culture preparation containing densely packed mitochondria has not been seen in situ, suggesting that changes in cell ultrastructure due to the conditions of culture cannot be discounted. However, the ultrastructure of the cultured cells was, for the most part, consistent with that of the same cell type in situ, indicating that the culture preparation may be a useful model for investigation of the roles and interactions of intramural neurones in the heart, which are inaccessible for such studies in situ.     

A proposal for a simple and inexpensive therapeutic cancer vaccine

In this essay, I propose a new method of treating tumours, using an old and inexpensive preparation, that I contend would be of considerable benefit to patients and their cancer management. My rationale for this treatment initially arose from recent advances in the understanding of dendritic cell function. (Dendritic cells are key cells of the immune system that are able to either turn on or turn off T-cell responses.) Evidence to support this approach is found in 100-year-old studies on the immunotherapy of cancer. Also, I draw on some remarkable, but little-known studies from the 1960s-1990s, demonstrating that the preparation has already been trialled in humans (although not intratumourally, as I propose), and is considered sufficiently safe to proceed with clinical trials in cancer volunteers.     

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System?

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues.     

Inhibitory effect of alpha-methyl-1-adamantane methylamine hydrochloride (rimantadine) on RNA-dependent RNA polymerase induction in culture of cells, infected with influenza virus]

An anti-influenza preparation, rimantadine (alpha-methyl-1-adamantane methylamine hydrochloride) at concentrations of 10--25 mkg/ml depresses the RNA-dependent RNA polymerase induction in a culture of cells infected with influenza virus (fowl plague virus). The inhibitory effect is also observed 2 hours following cell infection. In vitro studies have demonstrated that rimantadine has no effect on the activity of virus-induced RNA-dependent RNA polymerase, as well as on that of RNA-dependent RNA polymerase associated with virus particles.     

EPR characterization of an oxygen-evolving photosystem II preparation from the transformable cyanobacterium Synechocystis 6803.

The transformable cyanobacterium Synechocystis 6803 has a photosynthetic apparatus that is similar to that of plants. Because of the ease with which this organism can be genetically manipulated and isotopically labeled, Synechocystis has been used extensively in recent studies of electron transfer in the water-splitting complex, photosystem II. Here, we present the first EPR characterization of a highly active oxygen-evolving preparation from this organism. This preparation shows oxygen-evolution activities in the range from 2400-2600 mumol of O2/(mg of chlorophyll.h). We show that this preparation is stable enough for room temperature EPR studies. We then use this assay to show that the lineshapes of the D+ and Z+ tyrosine radicals are identical in this preparation, as has been observed in photosystem II complexes from a wide variety of photosynthetic species. We also present the first multiline EPR spectrum that has been observed from the Synechocystis manganese cluster.     

Isolated hepatocytes - past, present and future

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca2+-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase.The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.     

Evidence for Cell Surface Extracellular Matrix Binding Proteins in Hydra vulgaris

The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [³H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreerting studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-β₁ integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified two major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of >200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian     

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.     

A novel ‘pipeline’ system for downstream preparation of therapeutic monoclonal antibodies

A novel ‘pipeline’ system for the preparation of therapeutic monoclonal antibodies (mAb) in a non-GMP compliant environment has been developed. We used sterile silica-gel pipes to connect individual process units, in order to form a fully-enclosed and seamlessly connected system. This ‘pipeline’ system was used to implement downstream preparation processes for a humanized anti-CD146 mAb, huAA98, which is a therapeutic mAb generated to inhibit cancer-related angiogenesis. The quality assessment of the huAA98 end-product indicated that endotoxin levels were 0.016 EU/ml, protein A levels were 1.08 ng/ml and host cell protein (HCP) was undetectable. Thus, all measures were below the clinical criteria set by the Chinese Pharmacopoeia (Edition 2010). Having passed our proof-of-concept test, this ‘pipeline’ system can be used as a universal platform for the preparation of mAbs suitable for pre-clinical studies, in a non-GMP compliant laboratory environment.     

Platelet rich plasma in xeno-free stem cell culture: the impact of platelet count and processing method

Background: Stem cell culture for regenerative medicine needs platelet rich plasma (PRP) as fetal bovine/calf serum (FBS/FCS) substitute. However, the various studies used various protocols in preparing and processing the PRP. This study aimed to compare and conclude the most effective and efficient protocol. Methods: we searched in vitro studies that used human PRP as FBS/FCS substitute to culture human cells, and compared the various available protocols to identify the easiest and effective protocols for the preparation of PRP and the release of the growth factors (GFs) to support the highest cell growth in stem cell culture. Results: ten studies fulfilled the selection criteria and were included in the analysis. Discussion: Almost all studies on bone marrow mesenchymal stem cell (BM-MSC) and adipose stem cell (AT-SC) showed that platelet lysate and/or activated platelet releasate were superior or at least the same as either FBS or FCS, except for one study that got different results on human AT-SC. Several studies showed that either 5% activated PRP (aPRP) or platelet lysate (PL) was sufficient to support cell growth, or even better when they were compared to 10% FBS, while higher concentrations were counterproductive. However, some studies showed that 10% aPRP or PL was needed. The difference between studies was due to the difference in either the PRP preparation from blood and in the PRP processing to release the GFs, which yield various GF concentrations. Conclusion: In conclusion, studies are needed to reveal the optimal final platelet counts for the various PRP processing methods for various kinds of cells. The easiest PRP processing is freezing to -20?C followed by thawing, or thrombin activation using a final concentration of 100U/mL.     

A new method for the preparation of metaphase chromosomes for flow analysis

A new method for the preparation of metaphase chromosomes for flow analysis has been evaluated. It has been shown that this method, which involves detergent lysis of metaphase cells and polyamines to stabilize the DNA, yields lower coefficients of variation and background levels in the DNA histograms than is currently obtained by hexylene glycol based methods. A conventional flow cytometer (FACS-II) has been used to resolve the human karyotype into about 14 peaks after ethidium bromide staining and excitation with a relatively low level of illumination (0.4 W at 488 nm). Flow karyotypes have also been obtained from suspension cell lines, in particular from the mouse cell line, Friend 707/B10. The only disadvantage of this method is that the chromosomes are highly condensed and therefore banding studies on sorted chromosomes may not be possible.     

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

Biopharmaceutical manufacturing processes can be affected by variability in cell culture media, e.g. caused by raw material impurities. Although efforts have been made in industry and academia to characterize cell culture media and raw materials with advanced analytics, the process of industrial cell culture media preparation itself has not been reported so far. Within this publication, we first compare mid‐infrared and two‐dimensional fluorescence spectroscopy with respect to their suitability as online monitoring tools during cell culture media preparation, followed by a thorough assessment of the impact of preparation parameters on media quality. Through the application of spectroscopic methods, we can show that media variability and its corresponding root cause can be detected online during the preparation process. This methodology is a powerful tool to avoid batch failure and is a valuable technology for media troubleshooting activities. Moreover, in a design of experiments approach, including additional liquid chromatography–mass spectrometry analytics, it is shown that variable preparation parameters such as temperature, power input and preparation time can have a strong impact on the physico‐chemical composition of the media. The effect on cell culture process performance and product quality in subsequent fed‐batch processes was also investigated. The presented results reveal the need for online spectroscopic methods during the preparation process and show that media variability can already be introduced by variation in media preparation parameters, with a potential impact on scale‐up to a commercial manufacturing process.     

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Production of human immunodeficiency virus by chronically infected cells grown in protein-free medium.

A human T cell line chronically infected with Human Immunodeficiency Virus (HIV) has been adapted to grow in a chemically defined, protein-free medium. Virus particles are produced at rates comparable to those of serum-supplemented cultures; virus preparations free of undesirable proteins can be produced in preparative amounts by simple ultrafiltration procedures and cell culture supernatants can be used as such for the preparation of ELISA solid phases. This material has been used very conveniently for studies concerning characterization of antibodies against HlV-specific proteins, interaction of HIV with complement components and inclusion of human cell-derived proteins into virions; we propose its use as a powerful tool for the structural as well as functional analysis of the virus particle itself.