Multi-parametric flow cytometric cell cycle analysis using TO-PRO-3 iodide (TP3): Detailed protocols

TO-PRO-3 iodide (TP3), a monomeric cyanine nucleic acid stain with a peak absorbance at 642 nm and emission at 661 nm, is best excited by a helium-neon (HeNe) laser (633 nm). It was tested in monocytes and different cell lines under conditions of different fixatives, dye concentrations, labeling kinetics and RNAse concentrations for mono-, bi- and tri-parametric flow cytometric cell cycle analysis to establish the best protocol for DNA analysis in terms of G₁ peak CV, G₂/G₁ ratio and minimal amount of debris. A linear increase in G₁ peak position was found from 0.1 to 2 μM TP3 concentrations. Fixatives 70% ethanol or 1% methanol-free formaldehyde, followed by 70% ethanol, resulted in the best DNA histograms. Although different protocols were found to be cell-type specific, in general, excellent results were obtained with 30 min incubation with 0.5 μM TP3 plus RNAse in almost all cell lines tested. These data show that TP3 is an alternative method to propidium iodide (PI), the most commonly used DNA-specific probe in flow cytometry. The most important advantage of using TP3 in combination with other fluorochromes, such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in bi- or tri-parametric flow cytometric analysis, is that there is no need for fluorescence compensation for the TP3 signals.     

Flow cytometry and staining kinetics to monitor the G0/G1 transition

The phenanthridine dyes propidium iodide (PI) and ethidium bromide (EB) have contributed to make DNA quantitation a routine measurement by flow cytometry. As planar molecules they easily intercalate between base pairs of double stranded nucleic acids. When the quantity of dye exceeds the amount of DNA, the intensity of emitted fluorescence can be related to the amount of DNA. This is, in fact, the basic requirement of quantitative measurement. When staining is performed at low dye concentration and there are no sufficient molecules to saturate all the available DNA-binding sites, the emitted fluorescence, other than the true DNA quantity, may be dependent on the state of chromatin condensation. A staining procedure aimed to stress the influence of nuclear structure on the emitted fluorescence of PI is described. This is achieved by means of a very low dye concentration (<0.01 g/ml PI) to guarantee staining far below saturation condition. In this particular condition the staining rate slows down to be monitored by flow cytometry. As different experimental models the leucocytes of the normal human peripheral blood have been used taking into account the relatively condensed chromatin of granulocytes with respect to that of the mononuclear cells. Significantly higher fluorescence intensity have been obtained from mononuclear cells compared to that of the polymorphonuclear ones. Quiescent versus exponentially proliferating cell cultures had also been tested. In this staining condition low fluorescence intensity have been obtained by condensed chromatin structure (G₀) in comparison to the more decondensed (G₁) one.Abbreviations PI propidium iodide - PBS phosphate buffered saline - BrdU bromodeoxyuridyne - FCM flow cytometry - PMNC polymorphonuclear cells - MNC mononuclear cells     

Formation of the egg envelope of a teleost, Dicentrarchus labrax (L.): immunochemical and cytochemical detection of multiple components

The formation of the egg envelope in a teleost, Dicentrarchus labrax (L.), was analysed at histological and ultrastructural level. The sequential deposition of three main layers (Z1, Z2 and Z3) constitutes the extracellular matrix throughout oocyte development. Various findings indicate that these subunits are biochemically distinct: (1) periodate- and phosphotungstic acid-reactive carbohydrates are obviously detected only in the Z1, that constitutes the initial deposit of the egg envelope in early lipidic oocytes; (2) a monoclonal antibody (DLE7) against egg envelope polypeptides did not immunostain the Z1 and the underlying Z2; (3) the antigenic determinants recognised by DLE7, thought to be exogenous in origin (synthesised in the liver), are incorporated in the inner layer (Z3). In addition, DLE7 immunostained a thin layer, assembled together with Z3. This line has not yet been described in teleost eggs and was named Z1a. This study first describes at fine cytological level the contribution of exogenous proteins to formation of the different egg envelope layers. Results obtained with conventional, immunochemical and cytochemical techniques suggest multiple synthetic sources (exogenous and follicular) of egg envelope proteins.Abbreviations IR Immunoreactivity - LVS Lipid vesicle stage - mAb Monoclonal antibody - PAS Periodic acid–Schiff - PBS Phosphate-buffered saline - PTA Phosphotungstic acid - TBS Tris-buffered saline - TEM Transmission electron microscopy - YGS Yolk granule stage     

Effect of nitric oxide on lymphocytes from sporadic amyotrophic lateral sclerosis patients: toxic or protective role?

Abstract Markers of oxidative and nitrosative stress have been found in spinal cord, cortex, cerebrospinal fluid and plasma of patients affected by amyotrophic lateral sclerosis (ALS), a fatal disorder characterised by progressive motor neuron degeneration. In this study, we investigated the effect of the NO-releasing agent, diethylamine NONOate (NONO), on lymphocytes from patients affected by the sporadic form of ALS (SALS) and controls by flow cytometry. In the same experimental conditions we investigated the expression of the antioxidant proteins, Bcl-2 and SOD1. Incubation with NONO induced cell damage in control lymphocytes but did not further damage the already affected untreated SALS lymphocytes. The incubation with NONO induced a time-dependent decrease of Bcl-2 and SOD1 in control lymphocytes. Surprisingly, in SALS lymphocytes the NONO treatment increased the expression of these proteins, which in basal conditions was depressed compared to control lymphocytes.     

Motor and psychomotor functions in amyotrophic lateral sclerosis evaluated by tests of motor ability

18 patients with typical sporadic Amyotrophic Lateral Sclerosis (ALS) were investigated by the Motor Accuracy and Speed Test (MAST) and 18 healthy age-and-sex-matched volunteers, acted as controls. All performed each of the five tests 10 times with both the right and the left hands and repeated the experiment after one week by the same procedure. Motor performances were better in the controls than in the ALS patients only in the first three tasks. At retest, one week later, the controls generally improved while ALS patients did not. The mean percentages of changes showed a statistically significant difference in the fifth task. Analysis of the results suggests the possibility that, among the other mechanisms, a disturbance of motor learning ability could be operating in ALS patients.A preliminary communication upon this subject was given at the V International Congress on Neuromuscular Diseases, Marseille 12–18 September, 1982.     

Recombinant Semliki forest virus infects and kills human prostate cancer cell lines and prostatic duct epithelial cells ex vivo

Semliki Forest Virus (SFV) is a broad host range RNA virus capable of high-level recombinant protein expression and apoptosis induction in many cell types. We have successfully used a recombinant, replication deficient SFV vector to express the LacZ marker gene product in seven human prostate cell lines, as well as in human prostate tissue explants. Flow cytometry revealed that 40-60% of PPC-1 prostate cancer cells died 24-72 h after infection with SFV-LacZ virus. Most human prostate cancer cell lines expressed high levels of recombinant protein. Infection of human prostate tissue ex vivo led to similarly high expression levels but the recombinant beta-galactosidase was confined to duct epithelial cells. Infection of cell and tissue cultures resulted in detachment of adherent cells from the culture surface and detachment of epithelial cells from the basement membrane of tissue. Our results indicate that SFV may be useful in targeting recombinant protein expression and apoptosis to prostatic duct epithelial cells.     

Incorporation of the unusual C(alpha)-fluoroalkylamino acids into cyclopeptides: synthesis of arginine-glycine-aspartate (RGD) analogues and study of their conformational and biological behavior

A series of six arginine-glycine-aspartate (RGD) cyclopeptide analogues containing a C(alpha)-di- or trifluoromethylamino acid (alpha-Dfm or alpha-TfmAaa) at different positions of the ring were synthesized. All peptides were obtained in two diastereomeric forms, which were separated by HPLC. In vitro biological tests of the new cyclopeptides P were carried out in comparison with their corresponding cyclopeptides R lacking the alpha-fluoromethyl group. Five out of the six compounds P-I (containing (S)-alpha-Tfm-Aaa) showed activities in the nanomolar range, while the P-II compounds (containing (R)-alpha-Tfm-Aaa) were much less active or totally inactive. Only cyclo[RGDf-(S)-alphaTfmV] (P1-I) was found to be significantly more active than its model compound cyclo(RGDfV) (R1). The three-dimensional structure in water and DMSO was determined by NMR techniques and molecular dynamics (MD) calculations, but it was not possible to highlight significant differences in the backbone conformation of the peptides examined. Significant interproton distances, derived from nuclear Overhauser effect (NOE) experiments, were used to determine the absolute configuration of the side chains.     

Ovarian hyperstimulation-like syndrome after administration of triptorelin to a woman with endometriosis.

Ovarian hyperstimulation after a single dose of gonadotropin-releasing hormone (GnRH) analog is a rare phenomenon. A case of ovarian hyperstimulation-like syndrome after sole administration of triptorelin (Decapeptyl 3.75 mg) is reported in a woman who had undergone surgery for an endometriotic cyst. After administration of the drug, abdominal pressure increased with nausea and diffuse pelvic pain. Ultrasound examination showed bilateral enlargement of the ovaries (right 74 x 62 mm, left 62 x 53 mm), more than 10 follicles ranging in diameter from 15-25 mm, proliferative endometrium 7 mm thick and fluid in the Douglas pouch up to 25 x 23 mm thick. Estradiol plasma level was in the normal range. The syndrome spontaneously resolved in the course of treatment and a spontaneous pregnancy occurred when the triptorelin effect disappeared.