Applicability of differential fluorescein diacetate and propidium iodide fluorescence staining for monitoring algal growth and viability

Microalgae can be cultivated for producing high-valued products through the production of enzymes to offset the cost of CO_(2) sequestration,providing financial incentives.The viability of algae in the photobioreactor needs to be monitored to ensure biologically active live cells.In this study,we explored a simple fluorometry method for differentiation of live and dead algal cells in photobioreactors by fluorescein diacetate(FDA)and propidium iodide(PI)fluorescence staining.FDA stains fluorescent green to the living cells while PI stains the dead cells,allowing the discrimination of live and dead cells.The method was evaluated using two green algae and two strains of cyanobacteria grown in shake flasks and a continuously stirred photobioreactor.The method was found applicable for Chlorella pyrenoidosa and Synechococcus 7002 but was not applicable for the cultures of Scenedesmus dimorphus and Synechococcus elongatus 7942.We conclude that FDA is a good stain for monitoring live algal cells in photobioreactors but its applicability to individual species of algae must be evaluated.

Comparison of labeling methods and time course of traumatic brain injury-induced cell death in mice

BACKGROUND：Various molecular mechanisms of cell death following traumatic brain injury have been previously described.However,the time course of cell death remains unclear.TUNEL and Fluoro-Jade B labeling have been widely used to label apoptotic cells and neuronal degeneration.Propidium iodide （PI） functions as a biomarker of cell death in vivo.OBJECTIVE：To explore the role of PI labeling compared to TUNEL and Fluoro-Jade B staining for detecting neural cell death,and to observe time course of traumatic brain injury-induced cell death in mice.DESIGN,TIME AND SETTING：A randomized,controlled,animal experiment was performed at the Laboratory of Aging and Nervous Diseases,Soochow University from September 2007 to December 2008.MATERIALS：PI （B1221） was purchased from Sigma,USA.TUNEL kit was purchased from Roche Molecular Biochemicals,USA.Fluoro-Jade B was purchased from Chemicon,USA.METHODS：A total of 70 healthy,male,Kunming mice were randomly assigned to sham-surgery （n = 5） and model （n = 65） groups.Traumatic brain injury was established using the controlled cortical impact method.PI was intraperitoneally injected at 1 hour prior to animal sacrifice.MAIN OUTCOME MEASURES：TUNEL,Fluoro-Jade B,and Pl-positive cells were quantified using a double-labeling method to determine the time course of traumatic brain injury-induced cell death.RESULTS：PI labeled cells in an earlier phase of cell death than TUNEL and Fluoro-Jade B labeling.Pl-positive cells were observed immediately following injury,and the numbers rapidly increased in injured brain areas at 1 hour,peaked at 24-48 hours,and subsequently decreased at 3-21 days post-injury.TUNEL-labeled cells were significantly increased at 12 hours,while Fluoro-Jade B-labeled cells were increased at 6 hours after injury,with cells still visible at 6-48 hours post-injury.Moreover,a greater number of Pl-positive cells were observed compared to TUNEL- and Fluoro-Jade B-labeled cells.CONCLUSION：PI labeling is more sensitive and reliable than TUNEL and Fluoro-Jade B staining for detecting cell death following traumatic brain injury.Moreover,PI labeling can function as a reliable marker to estimate the entire time course of cell death.

Fluorometric Viability Assessment of Capacitated and Acrosome-Reacted Boar Spermatozoa by Flow Cytometry

Sperm capacitation involves functional changes, such as the removal or appearance of specific molecules and changes in the plasma membrane;the acrosome reaction (AR) is an exocytotic event induced by calcium influx, enabling the spermatozoa to penetrate the zona pellucida. These processes can be achieved only if the spermatozoa have good viability;indeed, determination of sperm viability is used for the assessment of semen quality. Membrane integrity and mitochondrial activity are important viability parameters of spermatozoa and fluorescent techniques based on membrane permeability to dyes have been developed to determine these parameters. The aim of this work was to determine the viability of boar sperm (fresh, one hour of capacitation induction and 20 min of AR induction) by flow cytometry using propidium iodide (PI) (1.25 μg/mL) and rhodamine 123 (R123) (0.20 μg/mL). Aliquots of 5 × 105 sperm were incubated with each fluorochrome separately and simultaneously for 10 or 20 min, respectively, at 38℃. The proportion of labeled spermatozoa and their fluorescence intensities were measured using a flow cytometer. The fluorescence index (FI) with PI gradually increased during the incubation and we found significant differences between all the groups. With R123, the FI increased in the capacitated sperm but decreased in the acrosome-reacted sperm, with significant differences between the fresh and capacitated spermatozoa. Our results suggest that the increase in the R123 fluorescence intensity in capacitated spermatozoa is due to changes in the mitochondrial membrane activity because the spermatozoa experienced changes in membrane fluidity and flagellar activation during capacitation. The use of fluorochromes and flow cytometry is a good tool for monitoring many markers of sperm function. Although capacitation and AR processes have been well studied, there is still much information to be elucidated with regard to these complex processes.

Correlation Analysis of the Results of Double Fluorescence(AO/PI) Staining and Clinical Outcomes

Objective To estimate the predictive value of double fluorescence [acridine orange （AO）/propidium iodide （PI）] staining results for fertilization rate and clinical outcomes and analyze the correlation between the results of AO/P1 staining and sperm apoptosis. Methods A prospective study was carried out, 235 infertile couples remedied using traditional in vitro fertilization （IVF） were included. Semen collected from 235 patients were stained by fluorescence dye, AO and PL at the same time the spermatozoa apoptosis rate was calculated by using flow cytometry to detect the rate of Annexin V＋/ PI- . The result of fluorescence was divided into 3 groups as green （G）, yellow （Y） and red （R） according to the color of fluorescence. The correlation between the percent of the 3 colors and clinical outcomes and spermatozoa apoptosis rate was evaluated. Results Significant negative correlation was observed between the percentage of Y and fertilization rate （r= -0.42, P=0.04）, no significant correlation was observed between the percentage of G, R and fertilization rate. The percentage of G, Y, R was not significantly different between pregnant patients and non-pregnant patients, respectiw,ly, while the percentage of Y was significant different between miscarriage patients and liveborn patients （r=0.6L P=0.01） and no significant difference exist in the percentage of G and R between liveborn patients and miscarriage patients. There was a significant positive correlation between the percentage of Y and the rate of Annexin V＋/ PI （r=0.53, P=0.04）, and no significant correlation was shown between the percentage of G, R and the rate of Annexin V＋/ PI-.Conclusion The percentage of yellow group of double fluorescence （AO/PI） staining will affect the fertilization rate and the miscarriage rate, and this group of spermatozoa may be connected with the spermatozoa apoptosis.