Flow cytometry (Biology)

Bovine adipose-derived stem cells were obtained from the subcutaneous abdominal adipose tissue. The cells were cultured by the modified tissue-explants method developed in our laboratory, and then analyzed using optical microscopy and flow cytometry. These cells were able to replicate in our cell culture conditions. cell Flow cytometry showed that bovine adipose-derived stem cells expressed mesenchymal stem cell markers (CD73 and CD90). Meanwhile, haematopoietic markers (CD45 and CD34) are absent from bovine adipose-derived stem cells. We also induced their adipogenic differentiation in vitro.

SUMMARY Flow cytometric analysis is used to evaluate various micro-organisms' susceptibility to antibacterial agents and disinfectants. This paper describes a study where Candida albicans was used to assess the method against three disinfectants. The disinfectants examined were: 70%-ethanol and two 70%-ethanol based commercial hand disinfectants with smoothing and cleaning additives. To detect the presence of damaged microbial cells EtBr was used. With the method, a 1 minute sample incubation time showed 36.25% ± 1.45 and 35.0% ±2,0 dead cells present as a result of commercial disinfectants-1 and-2, incubation; and 86.5% ±2.8 after the incubation with 70%-ethanol. With a 3-minute incubation time, the corresponding results were 43.25% ±3.95; 43.5% ±2.6; 86.5% ±4.3. These were significant in multi-group comparison. With a 5-minute incubation time, C. albicans samples showed an antiseptic activity against the chemicals under investigation of: 85.5% ±2.7; 84.5% ±2.4 and 91.3% ±3.57 respectively. Here there was no significant difference. RÉSUMÉ L'utilisation de la cytométrie de flux pour évaluer l'efficacité désinfection L'analyse par cytométrie de flux est utilisée pour évaluer la sensi-bilité de divers micro-organismes à des agents antibactériens et des désinfectants. Cet article décrit une étude où Candida albicans a été utilisé pour évaluer l´éfficacité de trois désinfectants. Les désinfec-tants examinés étaient les suivants: 70%-éthanol et deux 70% à base d'éthanol, désinfectants commerciaux pour les mains avec des additifs de lissage et de nettoyage. EtBr a été utilisé pour détecter la présence de cellules microbiennes endommagées. Avec ce procédé, après un temps d'incubation de 1 minute, il a été montré que 36,25% ± 1,45% et que 35,0 ± 2,0 de cellules mortes présentes avec les désinfectants commerciaux-1 et-2; et une valeur de 86,5% ± 2,8 après l'incubation avec 70%-éthanol. Avec un temps d'incubation de 3 minutes, les résultats correspondants étaient 43,25% ± 3,95; 43,5% ± 2,6; 86,5% ± 4,3. Ceux-ci étaient significatifs en comparaison multi-groupe. Avec un temps d'incuba-tion de 5 minutes, les échantillons C. albicans ont montré une activité antiseptique contre les produits chimiques sous enquête de: 85,5% ± 2,7; 84,5% ± 2,4 et 91,3 ± 3,57%, respectivement. Ici, il n'y avait pas de différence significative. Mots clés: cytométrie de flux, l'évaluation de microorganismes de sensibilité, désinfectant, la levure, la désinfection des mains

We look at the utility of flow cytometry in large- and small-scale population surveys of amphibians, work done to that point, and the implications for conservation projects ranging from the discovery of cryptic species to hybrid zones and toxicological impacts.

Using data gathered over a 4-year period from a large population of the A. laterale-jeffersonianum complex from Haliburton, Ontario, I examined four areas of reproductive ecology that may contribute to ecological partitioning: (1) fluctuating sex, hybrid, and ploidy ratios; (2) differential migratory response by triploid and tetraploid genomotypes; (3) ontogenetic differences among triploid and tetraploid genomotypes; and (4) genotypic differences within unisexual genomotypes. For each case I was able to reject a null hypothesis ascribing ecological equivalency to those biotypes involved. The most germane ecological differences among unisexuals were imbedded in the broad syndrome of delays in larval development, metamorphosis, and sexual maturity displayed by tetraploids. These findings parallel those reported in a large body of literature generated from laboratory studies of polyploid salamanders. Using these corresponding data, I conducted a broad review of potential developmental, physiological, and biotypic causes and consequences of the observed delays. The results indicated that several effects of polyploidy and hybridity directly influence community ecology and contribute to stable coexistence of bisexuals and unisexuals. Based on these findings, a strategic model summarizing interaction in these communities was constructed, from which testable hypotheses may be derived for future research. The findings of this study confirmed the ontological status of the biotypic distinctions (genomotype versus genotype) advanced here.

Due to recent advances in single cell genomics the sorting of single cells into 96-and 384-well plates is becoming increasingly popular. When sorting for single cell genomics the requirement is that the sorted cells are accurately and reproducibly deposited at the bottom of the PCR plate, in a very small volume (less than 10µl) of receiving liquid.

Aims:  HIV  infection  is  a  secondary  immunodeficiency  disorder
associated  with  loss  ofCD4+Tcells  and  immune  suppression.
Tregs  are  immune  suppressive  cells  and  involved  in  peripheral
tolerance.  Tregs  expressCD4
+
CD25
+
on  the  surface  and  Foxp3
+
internally.  Relative  frequency  of  Tregs  during  HIV/AIDS  is  not
fully known. CD4
+
CD25
+
Foxp3
+
Tcells (Tregs) play a vital role in
peripheral  tolerance  but  its  role  during  HIV/AIDS  is  not  fully
known.  Hence  this  study  was  conducted  with  the  aim  to
characterize the Tregs frequency among HIV patientsand healthy
controls.
Study design: Cross sectional study
Place  and  Duration  of  study: Department  of  Microbiology,
University  of  Madras,  chennai  and  Government  Hospital  Of
Thoracic Medicine, Chennai between October 2012 to  December
2013.
Methodology: 108  individuals  comprising  of  71  HIV  positive
patients (44 patients undergoing HAART and 27 without HAART)
and 37 were HIV negative healthy controls were enrolled in this
study.  PBMCs  were  isolated  and  stained  with  CD4/FITC,
CD25/APC, FoxP3/PE antibodies (BD Pharmingen) and analyzed
for Tregs frequency by flow cytometry (BD FACSCalibur).
Results:  Our  study  showed  that  healthy  controls  had  a  Tregs
frequency  of  4.1%.  When  Tregs  frequency  analysed  among
HIV/AIDS  patients  who  are  yet  to  receive  HAART  we  found  a
significant reduction in Tregs population i.e. 1.2%. Encouragingly
this deep plunge in Tregs population was found to be restored and
surged upon HAART upto 12.6%. There was no difference when
compared between gender or age or absolute CD4 count.
Conclusion:HAART is known to decrease viral load and improve
the  patient’s  CD4  cells  count.  Here  we  report  that  HAART  has
improved  not  only  the  CD4  cells  but  also  the  Tregs  frequency. This  study  add  one  more  dimention
to  Tregs  in  infectious  diseases
especially HIV/AIDS.

The successful performance of experiments is tightly related to the efficiency and reliability of the analytical methods used for the data analysis. This is particularly true when data is collected as images and the quantification of the biological effect behind the picture is often an issue. Consistent evaluation represents a challenge even for experienced biologists. Even though other techniques allow the quantification of fluorescent cell populations, (FACS, among others) image analysis is widely used. For this reason there is an increasing need of methods for reliable and reproducible quantification of images. To this aim a new algorithm for image analysis has been developed that allows rapid quantification of fluorescent Dual Reporter cells populations. These cells can express RFP (αSMA) or EGFP (collagen), and, via epi-fluorescence, come up as red or green, or yellow when RFP and GFP co-localize within the same cell. The algorithm can quantify and classify these subsets , by using a fuzzy, two-pass class partitioning, in addition to Difference of Gaussians and watershed segmentation techniques for blob detection and extraction. Results show average errors in the 2-6% range and an analysis time of 6-9 seconds per acquired image. The algorithm was validated against manual quantification techniques and compared to other state-of-the-art software. It allows rapid detection and extraction of Dual Reporter cells distributions in acquired epi-fluorescence optical fields. Finally, thanks to the implementation of semi-automated calibration procedures, the algorithm constitutes a totally objective instrument for quantification and classification measurements, especially when class boundaries are not sharply defined.

Radiation can produce biological damage, mainly oxidative stress, via production of free radicals, including reactive oxygen species (ROS). Nanoparticles are of interest as radioprotective agents, particularly due to their high solubility and bioavailability. Silymarin is a hepatoprotective agent but has poor oral bioavailability. Silymarin was formulated as a nanoemulsion with the aim of improving its bioavailability and therapeutic efficacy. In the present study, we evaluated self-nanoemulsifying drug delivery systems (SNEDDS) formulated with surfactants and co-surfactants. Nano-silymarin was characterized by estimating % transmittance, globule size, and polydispersity index, and by transmission electron microscopy (TEM). The nano-silymarin obtained was in the range of 3–8 nm diameter. With regard to DNA damage,measured by a plasmid relaxation assay, maximum protection was obtained at 10 µg/mL. Cytotoxicity of nano-silymarin to human embryonic kidney (HEK) cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against -radiation was assessed by reduction in micronucleus frequency and ROS generation, using the 2,7-dichlorodihydrofluoresceindiacetate (H2DCFDA) assay. Radiation-induced apoptosis was estimated by microscopic analysis and cell-cycle estimation. Nano-silymarin was radioprotective, supporting the possibility of developing new approaches to radiation protection via nanotechnology.

The objectives of the present study were to investigate the effects of three equilibration times (0, 2, and 4 h) and two extenders (TRIS or Bioxcell ® ) for cryopreservation of bull semen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine. There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. The control treatment (0 h equilibration) had the lowest values (P < 0.05) for total (MOT) and progressive motilities (PROG), and percentage of sperm with intact plasma and acrosomal membranes (IPIA), with no significant differences between extenders. Extender TRIS had greater cryoprotective action than Bioxcell ® , with greater MOT, PROG, IPIA at 2 and 4 h, as well as the lowest proportion of damaged plasma membrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4 h yielded the most desirable (P < 0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6% for 0, 2, and 4 h, respectively). Overall, the combination of TRIS and 4 h of equilibration was the most desirable semen cryopreservation method, with greatest MOT, PROG, and IPIA (TRIS-T4 = 26.8%; BIO-T4 = 18.3%) and the least DPM. In conclusion, based on objective analyses, equilibration during cryopreservation was essential for maintaining motility and integrity of sperm membranes; equilibration for 4 h yielded the greatest sperm survival, independent of the extender used.

a b s t r a c t
The objectives of the present study were to investigate the effects of three equilibration
times (0, 2, and 4 h) and two extenders (TRIS or Bioxcell®) for cryopreservation of bull
semen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine.
There were significant interactions between equilibration times and extenders for sperm
motility and membrane integrity. The control treatment (0 h equilibration) had the lowest
values (P < 0.05) for total (MOT) and progressive motilities (PROG), and percentage of
sperm with intact plasma and acrosomal membranes (IPIA), with no significant differences
between extenders. Extender TRIS had greater cryoprotective action than Bioxcell®, with
greater MOT, PROG, IPIA at 2 and 4 h, as well as the lowest proportion of damaged plasma
membrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4 h yielded the most desirable
(P < 0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6%
for 0, 2, and 4 h, respectively). Overall, the combination of TRIS and 4 h of equilibration
was the most desirable semen cryopreservation method, with greatest MOT, PROG, and
IPIA (TRIS-T4 = 26.8%; BIO-T4 = 18.3%) and the least DPM. In conclusion, based on objective
analyses, equilibration during cryopreservation was essential for maintaining motility and
integrity of sperm membranes; equilibration for 4 h yielded the greatest sperm survival,
independent of the extender used.

ABSTRACT: This study was performed to understand how the choice of cytotoxicity assay format affects the observed biocompatibility of nanosilver (nAg). nAg coatings are physical coatings containing silver (Ag) that have feature sizes of
100 nm or less, often in the form of nanoparticles or grains. They are used on medical devices to prevent infection, but in
spite of this intended benefit, observations of potential cytotoxicity from nAg have been reported in numerous published studies. For medical device regulation, cytotoxicity testing is part of a biocompatibility evaluation, in which specific test methods are chosen based on the technological characteristics and intended use of a device. For this study, nAg-coated tissue culture polystyrene surfaces were prepared using magnetron sputter coating, resulting in nAg films of 0.2 to
311 μg cm
2 Ag. These coatings exhibited nanometer-scale morphologies and demonstrated a > 4 log10 reduction in
Escherichia coli viability. It was observed that extracts of nAg caused no cytotoxicity to L929 mouse fibroblasts, but cells cultured directly on nAg coatings (direct-contact assay format) showed a dose-dependent reduction in viability by up to 100%
(P < 0.001). Results using inductively coupled plasma mass spectrometry to measure Ag release suggested that extracts of
nAg are not toxic because the dissolved Ag in those samples becomes less cytotoxic over time, probably owing to the reaction with cell culture media and serum (six-fold cytotoxicity reductions observed over a 24-h period). These findings highlight the potential value of direct-contact cytotoxicity testing for nAg in predicting biological interactions with cells or
tissue in vivo.

The mammalian cell entry (Mce) operon 3 (mce3) is one of four homologous mce operons of Mycobacterium tuberculosis, encoding six (Mce3A-F) invasin-like membrane-associated proteins. Previous studies have shown that recombinant expression of Mce1A encoded by the mce1 operon in Escherichia coli allows this non-pathogenic bacterium to invade and survive inside macrophages, and latex beads coated with Mce1A are internalized by non-phagocytic HeLa cells. However, the role of other mce1 operon proteins (Mce1B-F) and proteins encoded by the operons mce2-4 in facilitating the internalization of M. tuberculosis in mammalian cells has not been studied. This study was carried out to determine whether Mce proteins encoded by the mce3 operon also facilitated the internalization of latex beads by HeLa cells. Recombinant pure Mce3A and lipoprotein LprM (Mce3E) were expressed and purified from E. coli cells. Mce1A expressed as a fusion protein with glutathione S-transferase (GST-Mce1A) and GST alone, purified similarly from E. coli cells, were used as control proteins. Fluorescent latex beads coated with purified proteins were used to study their uptake by HeLa cells using fluorescence microscopy, flow cytometry and electron microscopy. Fluorescence microscopy and flow cytometry showed an association of HeLa cells with beads coated with both Mce3A and LprM, whilst GST-Mce1A and GST yielded the expected results. Transmission electron microscopy confirmed the uptake of beads coated with Mce3A or LprM by HeLa cells. The data showed that Mce3A encoded by the mce3 operon facilitated the uptake and internalization of latex beads by HeLa cells. The data also showed, for the first time, the role of another Mce protein (LprM/ Mce3E) in facilitating the interaction and internalization of M. tuberculosis by mammalian cells.

Autopentaploidy is a death sentence for vertebrate animals, which explains why none has ever been found outside of experimental laboratory settings. Allopentaplody appears to be somewhat better tolerated, with at least a few individuals able to make it past the embryonic stage and some to adulthood. Here, flow cytometry allows for documentation of the first known naturally occurring pentaploid vertebrate—a salamander with four sets of chromosomes from one species, and a single set from a related species.

Facilitated by the ability to screen large numbers of individuals via flow cytometry, this paper documents the results of the first large-scale ecological investigation of a bisexual-unisexual salamander community. Bisexual breeding dynamics largely conformed to the typical breeding dynamic within the genus, but our interesting findings with respect to hybrid migration timing had clear implications for breeding success, outcomes and ecological partitioning.

Immunological and structural characteristics of hemocyte populations in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae), going from two different Sicilian habitats (Faro Lake and Tyrrhenian sea), was investigated
by means of two different techniques (flow cytometric and micro-Raman spectroscopy analyses). For this purpose, three hundred and sixty mussels Mytilus galloprovincialis were analyzed during November 2017. They were
divided into two equal groups (triplicate sample) on the basis of the site of collection (n=60 caught in Faro Lake - group A, and n=60 caught in Tyrrhenian Sea - group B). Some several differences between the species of
Faro Lake and Tyrrhenian Sea are observed and ascribed to the disruption of immune parameters induced by the variations of some qualitative water parameters (temperature, salinity, dissolved oxygen, pH, ammonium 10,
free chlorine, total chlorine, total phosphate, orthofhosphate) recorded in the two habitats. This study is relevant for monitoring the conditions of the sea and Faro Lake, which is strongly influenced by the currents of the
Tyrrhenian Sea. Faro lake is well known for the cultivation of mussels and this is part of a coastal habitat of particular interest, consisted of a peculiar biocenotic complex. Further, for the first time, significant different
arrangement in the mussels cell structural organization was evidenced by simply following their highly reproducible Raman biomolecular signatures.

Life Sciences research at the level of individual cells is a rapidly developing research area. Traditional single-cell technologies such as cytometry and microscopy are being used in combination with the  emerging field of single-cell genomics.

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The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex max 646 nm; Em max 681 nm; Em range 665-Ͼ800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. Methods: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. Results: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared

Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows detection of intranuclear hTERT while maintaining identifiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by flow cytometry and telomerase activity detected by the telomeric repeat amplification protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations.

Docetaxel is a chemotherapeutic agent that is used against various cancers. However, it has several side effects due the solvent used in its formulation. In this study,the antitumor activity of docetaxel-loaded-microemulsion against HepG2 liver cancer cells was assessed by studying the cell toxicity and mechanism of cell death. The microemulsion formula consisted of weight percentages of 37 of cremophor/transcutol mixture (2:1, w/w), 29 of capryol 90 and 34 of water. Three microemulsion formulas were evaluated, freshly prepared docetaxel-loadedmicroemulsion (F-ME), stored formula of F-ME for two weeks (O-ME) and blank microemulsion formula (B-ME). The antiproliferative screening using sulphorhodamine B (SRB) assay revealed that the average cytotoxicities of all of the microemulsion formulations (F-ME, O-ME and B-ME) at all micromolar concentrations (1, 5 and 10) against HepG2 cells were more than Taxotere by 3 folds. The three formulas have induced apoptosis through altering the cell membrane in HepG2 cells as revealed by the images of ApopNexin FITC detection kit, light microscopy and transmission electron microscopy. Our findings demonstrated that blank microemulsion as well as the docetaxel-loaded microemulsions was having greater cytotoxicity than Taxotere against HepG2 cells.

We used flow cytometry to show that acutely sick or deformed green frogs, as well as apparently healthy individuals from the same agricultural settings in Quebec, displayed genomic disruptions consistent with exposure to pesticides, herbicides and other clastogenic compounds widely used in agriculture.