Development and application of a method for determination of activity and vitality of Hansenula polymorpha by carboxyfluorescein diacetate-propidium iodide dua fluorescent staining / 中国生物制品学杂志

@#Objective To develop a method for determination of activity and viability of Hansenula polymorpha by dual fluorescent staining with carboxyfluorescein diacetate(CFDA)and propidium iodide(PI).Methods The time durations(5,10,20,40,60 and 120 min for CFDA,5,10,15,20 and 25 min for PI)and working solution concentrations(50,100,200and 300 μg for CFDA,2,10,20 and 30 μmol/L for PI)for the dual fluorescent staining were optimized by single factor test. Under the optimal condition,the H.polymorpha samples at theoretical survival rates of 0,25%,50%,75% and 100%were determined,of which the fluorescent intensity was observed under fluorescent microscope,and the gray value was analyzed by ImageJ software. The live and dead cells were counted,based on which the actual survival and death rates were calculated. Meanwhile,the relationships of actual gray value,actual survival rate and actual death rate to the corresponding theoretical values were analyzed. Activity and viability of three batches of cultured H.polymorpha were detected by CFDA-PI dual fluorescence staining.Results The optimal time durations for staining with CFDA and PI were 60 and 5 min,while the optimal working solution concentrations were 200 and 2 μmol/L,respectively. The actual gray value,actual survival rate and actual death rate of H.polymorpha samples at various theoretical survival rates were significantly correlated to the corresponding theoretical values(R~2=0. 998 3～0. 999 2,P < 0. 05). The CVs of activity and viability values in three detections of three batches of H.polymorpha culture were 3. 20%～4. 03% and 1. 10%～2. 27%, respectively.Conclusion The CFDA-PI dual fluorescent staining was successfully developed,which may be used for determination of activity and vitality of H.polymorpha.

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

Antiproliferative effect of chitosan extracted from crab shells on human lung adenocarcinoma cell line (A549)

The Present study was made to evaluate the antiproliferative effect of chitosan extracted from crab shells against human lungadenocarcinoma cell line (A549). Chitosan was extracted from crab shells which includes deproteinization, demineralization,deacetylation. MTT method to find out the toxicity and cell viability of chitosan in both normal and cancer cells (A549). The Propidiumiodide staining and DNA fragmentation is to analyze the apoptotic bodies in A549 cell line. Chitosan appeared creamy white in colourand the total carbohydrate content was estimated as 0.07 mg/ml. The antiproliferative effect of chitosan against A549 cells clearlyemphasizes, that there is a decrease in the cell viability. The 50 % inhibition (IC50) of the cell growth was found at 20 µg/ml.The cytolocalization of nuclear morphology and DNA fragmentation assay revealed the induction of apoptotic cell death in A549 at 24 hours.Chitosan exhibits the inhibitory effect by inducing loss of cell viability, morphology change and DNA fragmentation in A549 cells due tothe presence of free protonated amino groups on the polymer chain. Our preliminary studies support that chitosan could be an efficienttherapeutic agent for cancer

Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage

Abstract INTRODUCTION Citronellal (Cit) possesses antifungal activity and has possible implications for reactive oxygen species (ROS) generation in Candida albicans. In this study, the effects of Cit on ROS generation and the mechanisms by which Cit exerts anti-Candida effects were examined. METHODS A 2′,7′-dichlorodihydrofluorescein diacetate assay was used to assess oxidative damage. Cell necrosis was determined by flow cytometry after FITC-Annexin V staining. Mitochondrial function was studied based on mitochondrial potential, metabolic activity (MTT assay), and phenotypic susceptibility on a non-fermentable carbon source. Membrane intactness and DNA damage were estimated by a propidium iodide (PI) uptake assay and 4',6-diamidino-2-phenylindole (DAPI) staining. RESULTS ROS generation was enhanced in response to Cit, leading to necrosis (2%). Additional hallmarks of cell death in response to Cit, such as mitochondrial membrane depolarization and DNA damage, were also observed. Cit treatment resulted in dysfunctional mitochondria, as evidenced by poor labeling with the mitochondrial membrane potential-sensitive probe rhodamine B, reduced metabolic activity (61.5%), and inhibited growth on a non-fermentable carbon source. Furthermore, Cit induced DNA damage based on DAPI staining. These phenotypes were reinforced by RT-PCR showing differences in gene expression (30-60%) between control and Cit-treated cells. Finally, PI uptake in the presence of sodium azide confirmed non-intact membranes and suggested that Cit activity is independent of the energy status of the cell. CONCLUSIONS Cit possesses dual anticandidal mechanisms, including membrane-disruptive and oxidative damage. Taken together, our data demonstrated that cit could be used as a prominent antifungal drug.

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95 percent ± 3.56, p < 0.0001, for 24 hours and mean of 37.67 percent ± 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.

Detection of Apoptosis by the Stainings of Annexin V, Propidium Iodide and Cytokeratin in OVCAR-3 Ovarian Cancer Cell Line / 대한산부인과학회잡지

OBJECTIVE: This study was designed to estimate the chemosensitivity by a quantitative evaluation of the apoptotic cell fractions using flow cytometry. METHODS: The OVCAR-3 cells were exposed to 20 nM or 30 nM taxol for 0 (control), 24 and 48 hours, then removed the taxol contained media, and cultured further with fresh media without taxol. (1) Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) were added to one test tube to detect the apoptotic cell fractions and at the same time, PI was added to the other tube to stain the DNA. (2) Annexin V-FITC and cytokeratin (clone CAM5.2 and MNF116) were added to the test tube. They were fixed and permeabilized with 1% paraformaldehyde solution and 100% methanol. They were then incubated with phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (GAM IgG1-PE or GAM IgG2a-PE) and sequentially stained with PI for DNA. All the stained cells were analyzed by a FACScan flow cytometer. RESULTS: (1) After treatment of 20 nM or 30 nM of taxol, G2M arrest was observed in both of treatment groups, which increased with time. (2) The G0G1 sub-fraction indicative of apoptosis increased with increase of culturing time from 24 hrs to 48 hrs. (3) The early apoptotic cell fraction with positive annexin V-FITC and negative PI increased with increase of culturing time. (4) In cells stained sequentailly with annexin V-FITC, cytokeratin (CAM5.2 and MNF116), and PI after 30 nM taxol treatment, the early apoptotic cell fractions increased with increase of culturing time. However, their extent was somewhat lower than those observed by positive annexin V-FITC and negative PI in cells treated with 20 nM of taxol. CONCLUSION: The results of sequential stainings with annexin V-FITC, cytokeratin, and PI were consistent with the those of annexin V-FITC and PI with parallel DNA staining. Our results suggested that the level of apoptosis detected by flow cytometry could be a marker of chemosensitivity which could select the sensitive anti-cancer agents before administration to gynecologic cancer patients.

Effects of Exposure of Propidium Iodide and Bisbenzimide on Differential Staining of Mouse Blastocysts / 대한불임학회지

OBJECTIVE: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. MATERIALS AND METHODS: A total 964 blastocysts (early~hatched) was exposed to PI (n=831) (group I:

A Rapid and Simple flow Cytometric Method for Measuring Cell Viability Using Propidium Iodide Staining and Forward Scatter Measurement

BACKGROUND: The importance of the determination of cell viability has prompted the development of several assays of viability that utilize the exclusion of certain dyes by viable cell membranes. Recently, flow cytometry has been adapted to estimate cell viability by using fluorescent dye which is excluded by living cells on the basis of altered dead cell properties. OBJECTIVE: We have developed a flow Cytometric method for measuring cell viability after staining with propidium iodide (PI) and have compared it with the classical colorimetric method, MTT assay, which is currently widely used in cytotoxicity assays in the research field. METHODS: We performed flow cytometry and MTT assay for the comparison of the sensitivity of the assessment of cell viability. RESULTS: Decrease of cell viability was measured by flow cytometry with the addition of as little as 0.002% Triton-X 100 in comparison to MTT assay which could only reveal a similar decrease of cell viability with the new method to 0.008% Triton-X 100. CONCLUSION: Our results demonstrate this new method to be more sensitive and simple for the assessment of cell viability.

INTRACEPHALIC DIFFUSION AND SELECTIVE LABELING OF PROPIDIUM IODIDE INJECTED INTO THE LATERAL CEREBRAL VENTRICLE / 解剖学报

Injections of propidium iedide (PI) into the lateral cerebral ventricle (LV) ofthe rat resulted in a prominent abnormality characterized by tremor,ataxia,andnystagmus.The intensity of PI fluorescence in the parenchyma of the brain fadedgradually away from the injection site and ventricles to the surfaces of the brain. In the forebrain it was seen that PI fluorescence reached the most lateral part ofthe ipsilateral caudate putamen nucleus.A constant neuronal labeling was observedin the septohippocampal nuclei,the A8-9-10 dopaminergic cell groups of themidbrain,the dorsal raphe nucleus,the median raphe nucleus,neurons within anddorsal to the medial lemniscus of the caudal midbrain,and Furkinje cells of thecerebellum.This neuronal labeling was bilateral.No distinct labeling was seen inother areas of the brain.Combined with Faglu histofluorescence,it was found thatalmost all of the dopaminergic neurons in the midbrain exhibited PI fluorescence.No labeled non-dopaminergic neuron was seen in A8-9-10.With a transection ofthe unilateral medial forebrain bundle,a prominent accumulation of PI fluorescencewas seen within the distal segments of catecholaminergic fibers near the transection,but no accumulation of PI was seen in the proximal segments.With LV injectionof Evans blue(EB)or DAPI or ethidium bromide,animals did not exhibit anyvisible abnormality.In animals with LV injection of EB or DAPI,although somelabeled cells were seen in the distant areas of the brain,their distribution wasdistinctly different from that of PI labeling.The above results indicate that besidesconfirming the LV injection of PI results in a prominent abnormality and PI isselectively uptaken by Purkinje cells,we have found that:a)PI is able to enter theparenchyma from the cerebrospinal fluid and diffuse widely in the brain;b)LVinjection of PI results in a selective labeling in certain specific areas of the brain,and those selectively labeled cells in A8-9-10 all are dopaminergic neurons;c)these dopaminergi(?) cells are labeled through axonal uptake and retrograde transportof PI.