Effects of UVB irradiation on pig epidermal cell kinetics in vivo: A DNA-flow cytometric analysis

Health hazards of titanium dioxide nanoparticles (TiO2-NPs) have raised severe concerns because of the paucity of information regarding the toxic effects among the population. In the present research, the in vitro and in vivo cytotoxic potential of TiO2-NPs were evaluated using flow cytometric techniques. Further, in vitro and in vivo genotoxic endpoints were estimated by means of comet, micronucleus (MN), and chromosomal aberration (CA) assays. In vitro analysis was performed at the concentration range of 10–100 µg/mL using murine RAW 264.7 cells. In vivo experiments were conducted on Albino mice (M/F) by exposing them to 200 and 500 mg/kg TiO2-NPs for 90 days. Decreased percentage of cell viability with higher doses of TiO2-NPs was evident in both in vitro and in vivo flow cytometric analysis. Further, an impaired cell cycle (G0/G1, S, and G2/M) was reflected in the present investigation following the exposure to TiO2-NPs. Increased comet scores such as tail length, % DNA in tail, tail moment, and olive moment were also observed with the higher doses of TiO2-NPs in vitro and in vivo comet assays. Finally, the in vivo MN and CA assays revealed the formation of MN and chromosomal breakage following the exposure to TiO2-NPs.

Download Full-text

329 FLOW CYTOMETRIC ANALYSIS OF SPERMATOZOA FROM REPORTER TRANSGENIC BOARS DERIVED BY PRECISION GENETIC ENGINEERING

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab329 ◽

2013 ◽

Vol 25 (1) ◽

pp. 312

Author(s):

W. Garrels ◽

S. Holler ◽

N. Cleve ◽

S. Klein ◽

Z. Ivics ◽

...

Keyword(s):

Germ Line ◽

Flow Cytometric Analysis ◽

Embryonic Stem ◽

Sleeping Beauty ◽

Large Animal Model ◽

Large Animal ◽

Reporter Protein ◽

Cassette Exchange ◽

Flow Cytometric

Recently, we produced 2 founder boars with a non-autonomous Sleeping Beauty (SB) system carrying 3 monomeric integrations of a Venus transposon cassette and showing transgene segregation during meiosis (Garrels et al. 2011 PLoS One 6, e27563). It was possible to show transmission of the reporter protein to fertilized oocytes by confocal microscopy. The aim of this study was to assess the suitability of different fluorophore reporters for in vivo labelling of pig spermatozoa. Therefore, we used Venus transposon fibroblasts from a F1 boar, which carry a single integration of the transposon cassette and used these fibroblasts for a Cre-mediated cassette exchange against an mCherry reporter. These cells were used for somatic cell nuclear transfer (SCNT) to derive a syngene clone cohort of boars, which differ only in the fluorophore reporter cDNAs (either Venus or mCherry). Importantly, this methodology did not request any antibiotic selection cassette and allows precise genetic modifications in a livestock species where no authentic embryonic stem cells are available (Garrels et al. 2012 Trends in Biotechnology 30, 386–393). A total of 8 male piglets carrying the Venus transposon, and 4 male piglets carrying the mCherry reporter were born. Three Venus boars and 2 mCherry boars were raised to sexual maturity, and ejaculated sperm was obtained with the help of a phantom. A detailed flow cytometric analysis revealed that the spermatozoa samples were specifically Venus or mCherry positive [Gallios, Beckmann Coulter, Krefeld, Germany; solid-state laser (488 nm; 22 mW), filter for green fluorescence (525 BP); filter for red fluorescence: (620/30)], respectively. In direct comparative measurements, the spermatozoa samples from transgenic boars (Venus and Cherry) and wildtype controls could be discriminated. Interestingly, spermatozoa were uniformly Venus- or mCherry-positive and gave a distinct fluorescence peak in flow-cytometric measurements. The monomeric transgenes were transmitted through the germ line according to Mendelian rules with the expected ratio of 50% transgenic and 50% nontransgenic piglets. Fluorescence microscopic analysis and Western blotting confirmed the uniform presence of Venus and mCherry in boar spermatozoa, respectively. This is the first characterisation of spermatozoa from a pig cohort carrying a targeted cassette exchange. This large animal model may help to elucidate the function of paternally transmitted components to fertilized oocytes.

Download Full-text

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽

10.1182/blood.v95.3.855.003k41_855_862 ◽

2000 ◽

Vol 95 (3) ◽

pp. 855-862 ◽

Cited By ~ 82

Author(s):

Robert A. J. Oostendorp ◽

Julie Audet ◽

Connie J. Eaves

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Flow Cytometric ◽

Differentiation Status ◽

Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

Download Full-text

Proliferative Tumor Doubling Times of Prostatic Carcinoma

Prostate Cancer ◽

10.1155/2011/301850 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Priya N. Werahera ◽

L. Michael Glode ◽

Francisco G. La Rosa ◽

M. Scott Lucia ◽

E. David Crawford ◽

...

Keyword(s):

Normal Tissue ◽

Prostatic Carcinoma ◽

Flow Cytometric Analysis ◽

Flow Cytometric Assay ◽

Flow Cytometric ◽

Biological Potential ◽

Aggressive Tumors ◽

Doubling Times ◽

Biological Differences

Prostate cancer (PCa) has a variable biology ranging from latent cancer to extremely aggressive tumors. Proliferative activities of cancers may indicate their biological potential. A flow cytometric assay to calculate maximum proliferative doubling times (Tmax) of PCa in radical prostatectomy specimens after preoperativein vivobromodeoxyuridine (BrdU) infusion is presented. Only 4/17 specimens had tumors large enough for flow cytometric analysis. TheTmaxof tumors was similar and ranged from 0.6 to 3.6 months. Tumors had calculated doubling times 2- to 25-fold faster than their matched normal tissue. Variations in labeling index andTmaxwere observed within a tumor as well as between different Gleason grades. The observed PSA doubling times (PSA-DT) ranged from 18.4 to 32.0 months, considerably slower than the correspondingTmaxof tumors involved. While lack of data for apoptotic rates is a limitation, apparent biological differences between latent versus aggressive PCa may be attributable to variations in apoptotic rates of these tumors rather than their cell proliferative rates.

Download Full-text