Evaluation of sperm mitochondrial function using rh123/PI dual fluorescent staining in asthenospermia and oligoasthenozoospermia

Objective：The recent advent of flow cytometry（FCM）,coupled with fluorescent dyes,has been successfully applied to assess mitochondrial function.The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium（Rh123/PI）dual fluorescent staining and FCM in asthenospermia and oligoasthenozoospermia.Methods：Twenty-five fertile men（with normal sperm parameters）and 230 infertile patients were examined.Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups：asthenospermia（n=30）and oligoasthenozoospermia（n=25）.Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function.Results：Significant differences were found between the normal and abnormal semen samples（P0.05）when Rh123＋/PI-,Rh123-/PI＋and Rh123-/PI-sperm were examined by FCM,but there was no significant difference between the asthenospermia（P=0.469） and oligoasthenozoospermia group（P=0.950）when Rh123＋/PI-and Rh123-/PI＋sperm were then examined;however,a significant difference was found between the 2 groups（P=0.003）when Rh123-/PI-sperm were examined.There was no correlation between Rh123-/PI-sperm and semen parameters in the normal group,but there was a significant negative correlation between the sperm concentration and Rh123-/PI-sperm in asthenospermia and oligoasthenozoospermia patients（r=-0.509,-0.660;P=0.018,0.038）.Conclusion：Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mitochondrial membrane potential in asthenospermia and oligoasthenozoospermia.

Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion

DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.

Experimental evaluation of fluorescent (alizarin red S and calcein) and clip-tag markers for stock assessment of ark shell, Anadara broughtonii

Release programs to enhance stocks of ark shell(Anadara broughtonii) have been undertaken in a number of Asian countries,but their effectiveness has rarely been investigated owing to a lack of marking methods.The quality and longevity of fluorescent markers,alizarin red S(ARS) and calcein(CAL)(200 and 300 mg/L),as well as clip tags,were tested on juvenile A.broughtonii.No significant differences in survival or shell growth were observed in juveniles stained with either of the two fluorochromes after a 160-day culture period,but the retention rate was 100%after 1 year.Fluorescent marks(>grade 3) were observable microscopically in juveniles stained with the two fluorochromes,and some fluorescent marks(>grade 4) were visible with the naked eye after 1 year.ARS-marked shells were brighter than those marked with CAL,and shells marked with 300 mg/L of the fluorochromes were easier to detect than those marked with 200 mg/L.Clip tags were incorporated into the shell as the bivalve grew,and the retention rate was64.25%after 160 days.Significant differences in survival(at 30 days),shell length(at 60,90,120,and 160days),and wet weight(at 90,120,and 160 days) were observed between the clip-tagged and control groups(all P<0.05),indicating that the tags may have passive effects on the ark shell.The results suggest that both ARS and CAL are suitable to mark A.broughtonii for large-scale restocking programs,and that optimal marking quality was achieved with 300 mg/L ARS.Lighter and smaller clip tags need to be developed to reduce injury and increase survival rate of clams.

Mouse Karyotype Obtained by Combining DAPI Staining with Image Analysis

In this study, mitotic metaphase chromosomes in mouse were identified by a new chromosome fluorescence banding technique combining DAPI staining with image analysis. Clear 4＇, 6-diamidino-2-phenylindole （DAPI） multiple bands like （J-hands could be produced in mouse. The Meta- Morph software was then used to generate linescans of pixel intensity for the banded chromosomes from short arm to long arm. These linescans were sufficient not only to identify each individual chromosome but also analyze the physical sites of bands in chromosome. Based on the results, the clear and accurate karyotype of mouse metaphase chromosomes was established. The technique is therefore considered to he a new method for cytological studies of mouse.

Detection of Mycoplasma Contamination in Animal Cell Cultures

[ Objective ] To develop a rapid efficient method for detecting mycoplasma contamination in cell cultures. [ Method] A pair of primers was designed according to two highly conserved nucleotide sequences of the 16S RNA from six kinds of mycoplasma that commonly contaminated cells. Then the mycoplasma contamination of 25 cell samples was defected by PCR and DNA fluorescence staining. EResultl When these cell samples were detected by DNA fluorescence staining, the positive rate and probable positive rate were respectively 24% and 16%. And when they were detected by PCR, the positive rate was 36%. [ Condusion] The PCR method is more sensitive and specific than the DNA fluorescence staining, and combining these two methods is the optimal way to detect mycoplasma contamination in cell cultures.

Applicability of LIVE/DEAD BacLight stain with glutaraldehyde fixation for the measurement of bacterial abundance and viability in rainwater

Rainwater contains substantial bacteria and rain is an efficient pathway for the dissemination of bacteria from the atmosphere to land and water surfaces.However,quantitative information on rainwater bacteria is very limited due to the lack of a reliable method.In this study,the epifluorescence microscopy enumeration with the LIVE/DEAD BacLight Bacterial Viability Kit stain was verified to quantify the abundance of viable and non-viable bacterial cells in rainwater,with the 4＇,6-diamidino-2-phenylindole（DAPI） stain for the reference of total cell counts.Results showed that the total counts of bacterial cells by LIVE/DEAD BacLight staining were consistent with those by DAPI staining,and the average detection efficiency was（109 ± 29）%.The ratio of cell count with glutaraldehyde fixation to that without fixation was（106 ± 5）%on average.The bacterial concentration in negative control was usually an order of magnitude lower than that in rainwater samples.However,in case of small precipitation,the abundance in negative control could be more than that in rainwater samples.These results indicate that the enumeration with LIVE/DEAD BacLight bacterial viability assay coupled with glutaraldehyde fixation and careful negative control investigation is an approach applicable to the measurement of the concentration and viability of bacterial cells in rainwater.

Exploring three-dimensional biological tissues with new chemical labels

Three-dimensional(3D)histology has exhibited tremendous potential in fundamental research and clini-cal disease grading,but compatible labeling techniques are still lacking.Recently in Science Advances,Pac et al.report a new histological technique termed 3DNFC,which realizes 3D fluorescence imaging of thick tissues via citrate-based in situ fluorophore formation.

Laser textured dimple-patterns to govern the surface wettability of superhydrophobic aluminum plates

A laser texturing technique herein can endow bare aluminum alloy surface with regular dimple-pattern array and thus generates a case hardening.After STA treatment,these laser-textured samples become superhydrophobic.The surface wettability of the laser-textured samples can be regulated by controlling the dimple-pattern dimensions during the laser processing.It is noteworthy that a fluorescence method is utilized to record the zones on the superhydrophobic surface penetrated by small enough water molecules.Compared with a general method of the Cassie-Baxter theoretical calculation,this fluorescence method intuitively exhibits the air trapping ability of the superhydrophobic surface.Furthermore,the laser-textured superhydrophobic samples have a notable hysteresis phenomenon at the initial period of UMT friction because the air cushion trapped within superhydrophobic samples have strong repellency against water droplets on the hydrophilic steel ball.Additionally,such samples display strong mechanical stability in comparison with bare aluminum alloy because of the presence of case hardening on the surface of the laser patterns.The research results above provide a valuable reference for designing a surface with different wettability,which may inspire practical applications in the fields of fluid transport,droplet manipulation,water harvesting and microfluidic devices.