scholarly journals Qualitative Assessment of Islet Viability by Staining with Fluorescein Diacetate (FDA) and Propidium Iodide (PI) Dyes v1 (protocols.io.bppwmmpe)

protocols.io ◽  
2020 ◽  
Author(s):  
Human Islet
Keyword(s):  
Propidium Iodide ◽  
Fluorescein Diacetate ◽  
Qualitative Assessment

A comparison of fluorescein diacetate and propidium iodide staining andin vitroexcystation for determiningGiardia intestinaliscyst viability

Parasitology ◽  
1989 ◽  
Vol 99 (3) ◽  
pp. 329-331 ◽  
Author(s):  
A. L. Smith ◽  
H. V. Smith
Keyword(s):  
Lower Limit ◽  
Propidium Iodide ◽  
Giardia Intestinalis ◽  
Fluorescein Diacetate ◽  
Fluorescent Indicators ◽  
Vital Dyes ◽  
Propidium Iodide Staining ◽  
Cyst Viability

SummaryThe viability of 4 human isolates ofGiardia intestinaliscysts using either the fluorogenic vital dyes fluorescein diacetate (FDA) and propidium iodide (PI) orin vitroexcystation was assessed. Whereas viable cysts, as defined byin vitroexcystation were present in each of the 4 isolates, cysts from only 3 of the 4 isolates took up the vital dyes. FDA consistently over-estimated cyst viability whilst PI under-estimated non-viable cysts when compared within vitroexcystation. Followingin vitroexcystation, both FDA and PI stained a proportion of unexcysted cysts indicating that FDA stained cysts which were incapable of excystation, whereas PI did not stain all cysts which were incapable of excystation. One human cyst isolate, which underwentin vitroexcystation, could not be stained with either FDA or PI. In the absence of currently more specific fluorescent indicators of viability, PI alone could be used to determine the lower limit of nonviability in positive water-related samples, where small numbers of cysts are to be expected.

A 3D environment for anulus fibrosus regeneration

2012 ◽  
Vol 17 (2) ◽  
pp. 177-183 ◽  
Author(s):  
Mario Cabraja ◽  
Michaela Endres ◽  
Aldemar A. Hegewald ◽  
Samuel Vetterlein ◽  
Claudius Thomé ◽  
...  
Keyword(s):  
Human Serum ◽  
Propidium Iodide ◽  
Polyglycolic Acid ◽  
Intervertebral Discs ◽  
Fluorescein Diacetate ◽  
Marker Genes ◽  
Anulus Fibrosus ◽  
Relieve Pain ◽  
3D Assembly

Object Biological repair strategies for the treatment of degenerated intervertebral discs are of growing interest. In addition to the development of nucleus pulposus implants to restore disc height and relieve pain, there is growing demand for an appropriate method for reconstructing the anulus fibrosus (AF). The aim of this pilot study was to evaluate the applicability of a resorbable 3D polymer of pure polyglycolic acid (PGA) combined with hyaluronan for the use in cell-free and cell-based regeneration and repair of the AF. Methods Adult human AF cells were expanded in vitro using human serum and rearranged three dimensionally in hyaluronan-PGA scaffolds that were stabilized with fibrin for in vitro analyses. The capacity of dedifferentiated AF cells to redifferentiate was evaluated after 2 weeks of culture, using propidium iodide/fluorescein diacetate staining, gene expression analysis of typical marker genes, and histological staining of proteoglycans. Results The propidium iodide/fluorescein diacetate staining demonstrated that vital human AF cells were evenly distributed within the construct. The induction of typical AF marker genes such as collagen Types I–III indicated the initiation of AF redifferentiation by 3D assembly in hyaluronan-PGA. Histological analysis of the constructs showed initial formation of an AF-like matrix comprising proteoglycans. Conclusions The results suggest that the 3D arrangement of human AF cells in resorbable hyaluronan-PGA scaffolds cultured in the presence of human serum is an excellent system for AF cell redifferentiation.

scholarly journals Cryptosporidium parvum metalloaminopeptidase inhibitors prevent in vitro excystation.

1996 ◽  
Vol 40 (12) ◽  
pp. 2781-2784 ◽  
Author(s):  
P C Okhuysen ◽  
C L Chappell ◽  
C Kettner ◽  
C R Sterling
Keyword(s):  
Enzymatic Activity ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Chelating Agents ◽  
Fluorescein Diacetate ◽  
Dye Uptake ◽  
Enzymatic Assays ◽  
Arginine Aminopeptidase

Cryptosporidium parvum arginine aminopeptidase (RAP) was studied during in vitro excystation. Specific RAP inhibitors were identified by using C. parvum extracts. Amastatin, a series of alpha-aminoboronic acids, and the chelating agents EDTA and 1,10-phenanthrolene, but not endoproteinase inhibitors, blocked enzymatic activity. RAP inhibitors found to be effective in soluble enzymatic assays were then studied for their effect on in vitro excystation. 1,10-Phenanthrolene, amastatin, and H-boronorleucine (pinacol) inhibited excystation by 84, 57, and 61%, respectively, compared with solvent-treated control oocysts. Sporozoites remained viable within the oocyst as determined by propidium iodide and fluorescein diacetate dye uptake, suggesting that alpha-aminoboronic acids were not directly lethal to the parasite.

scholarly journals An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide.

1985 ◽  
Vol 33 (1) ◽  
pp. 77-79 ◽  
Author(s):  
K H Jones ◽  
J A Senft
Keyword(s):  
Cell Viability ◽  
Propidium Iodide ◽  
Accurate Estimate ◽  
Fluorescein Diacetate ◽  
Staining Procedure ◽  
Blue Dye ◽  
Double Staining ◽  
Viable Cells ◽  
Bright Green

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.

270 PRETREATMENTS OF PORCINE SPERM BEFORE INTRACYTOPLASMIC SPERM INJECTION AFFECT QUANTITY OF PLCZETA;

2009 ◽  
Vol 21 (1) ◽  
pp. 232
Author(s):  
M. Nakai ◽  
J. Ito ◽  
K. Sato ◽  
J. Noguchi ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Propidium Iodide ◽  
Intracytoplasmic Sperm Injection ◽  
Blastocyst Stage ◽  
Fluorescein Diacetate ◽  
Control Group ◽  
Oocyte Activation ◽  
Group 2 ◽  
Almost All ◽  
Triton X

In pigs, the intracytoplasmic sperm injection (ICSI) procedure alone is insufficient to induce oocyte activation for embryonic development. Artificial activation can be accomplished, such as by electrical pulse-enhanced in vitro development to the blastocyst stage (Nakai et al. 2006). It is well known that the sperm factor (phospholipase Cζ; PLCζ) in spermatozoa, which triggers oocyte activation, is diffused into ooplasm when sperm fuse with oocytes. Our previous study showed that the activation rate of porcine oocytes injected with one sonicated sperm head was significantly lower than that of oocytes injected with a whole spermatozoon or with 3 sonicated sperm heads (Nakai et al. IETS 2007). These results suggest that the sonication treatment per se may affect the quantity of PLCζ in sperm. Furthermore, various pretreatments of sperm besides sonication have been conducted (e.g. removal of the sperm membrane) to increase the efficacy of ICSI. In this study, we investigated the effect of pretreatments (sonication, Triton X-100, and repeated cycles of freezing–thawing without cryoprotectant) on the quantity of PLCζ in porcine sperm. Cryopreserved-thawed boar-ejaculated sperm were used for 3 experimental groups: (1) sperm were sonicated for 10 s in pig-fertilization medium (pig-FM; Suzuki et al. 2002; Soni group), (2) freezing–thawing was repeated 3 times in pig-FM without cryoprotectant (3-F/T group), or (3) sperm were incubated in pig-FM supplemented with 0.1 or 1% Triton X-100 at 37°C for 1 min (0.1 and 1% Triton X-100 groups, respectively). Cryopreserved-thawed whole sperm without any treatment was used as a control. Results from staining with fluorescein diacetate and propidium iodide showed that almost all sperm were propidium iodide positive (dead sperm) immediately after the each treatment. In the control group, approximately 40% of sperm were fluorescein diacetate positive (live sperm) after thawing. The presence of PLCζ (72 kDa) was examined by Western blotting using the antibody against the N-terminal 19-mer sequence of porcine PLCζ (Kurokawa et al. 2005). A band corresponding to porcine PLCζ was not detected in any treatment group in any culture period (from 0 to 135 min). In contrast, PLCζ was detected in the control group and in all culture periods. These results strongly suggest that PLCζ in porcine sperm was lost immediately after the pretreatments, such as by sonication, incubation with 0.1 or 1% Triton X-100, and repeated cycles of freezing–thawing. The decrease in PLCζ protein by pretreatment may be one of the causes of incomplete activation of oocytes in porcine ICSI. This work was supported by a Grant-in-Aid for JSPS Fellows.

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