Propidium Iodide Nucleic Acid Stain in Euplotes v1

protocols.io ◽  
2017 ◽  
Author(s):  
Angela Piersanti
Keyword(s):  
Nucleic Acid ◽  
Propidium Iodide ◽  
Nucleic Acid Stain

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

Reproduction ◽  
2000 ◽  
pp. 145-152 ◽  
Author(s):  
B Pintado ◽  
J de la Fuente ◽  
ER Roldan
Keyword(s):  
Nucleic Acid ◽  
Propidium Iodide ◽  
Hoechst 33258 ◽  
Experimental Conditions ◽  
Functional Studies ◽  
Viable Cells ◽  
Direct Microscopic Examination ◽  
Acrosomal Exocytosis ◽  
Viable Sperm ◽  
Boar Spermatozoa

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.

Underestimation of bacterial numbers in starvation-survival mode using the nucleic acid stain DAPI

2003 ◽  
Vol 157 (3) ◽  
pp. 309-319 ◽  
Author(s):  
Christopher J. McNamara ◽  
Michael J. Lemke ◽  
Laura G. Leff
Keyword(s):  
Nucleic Acid ◽  
Starvation Survival ◽  
Nucleic Acid Stain ◽  
Survival Mode

Flow Cytometry to EvaluateAnaplasma marginaleParasitemia Using a Fluorescent Nucleic Acid Stain

2008 ◽  
Vol 1149 (1) ◽  
pp. 111-113 ◽  
Author(s):  
Rosalía Moretta ◽  
Paula Ruybal ◽  
María Mesplet ◽  
Romina Petrigh ◽  
Pablo Nuñez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Nucleic Acid Stain

Effect of nucleic acid stain Syto9 on nascent biofilm architecture of Acinetobacter sp. BD413

2005 ◽  
Vol 52 (7) ◽  
pp. 195-202 ◽  
Author(s):  
R. GrayMerod ◽  
L. Hendrickx ◽  
L.N. Mueller ◽  
J.B. Xavier ◽  
S. Wuertz
Keyword(s):  
Nucleic Acid ◽  
Microbial Population ◽  
Separate Flow ◽  
Flow Cell ◽  
Cellular Level ◽  
Flow Cells ◽  
Biofilm Architecture ◽  
Acinetobacter Sp ◽  
Nucleic Acid Stain ◽  
Architecture Development

Flow cells were utilized to determine the effects of repetitive Syto9 staining on developing Acinetobacter sp. BD413 biofilm and to identify features describing reproducible biofilm architecture at 63× magnification. Syto9 is a general nucleic acid stain employed to visualize the entire microbial population of the biofilm and a component in the LIVE/DEAD® BacLight™ Bacterial Viability kits. CLSM images were quantified with the biofilm analysis software PHLIP to calculate six commonly used biofilm architecture characteristics. The characteristics biovolume and mean thickness were most reproducible when biofilms were grown in separate flow cells under controlled conditions, while roughness, porosity, total spreading and surface area to biovolume ratio exhibited inherent variability. Biovolume was more variable in separate flow cells than in channels of the same flow cell. However, even biofilms grown in channels of the same flow cell did not generate reproducible architectures based on the six characteristics. Results suggest difficulties in differentiating the effect of changes due to treatment from the natural variability of architecture development at the cellular level. Despite this high variability, biofilms only stained once developed into thicker structures containing more biomass than biofilms stained multiple times, suggesting that repeated staining with Syto9 affects architecture development. The application of Syto9 to monitor developing biofilms is not recommended.

Novel Reagents for the Addition of Viability Measurements to Immunostaining Using Flow Cytometry.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3879-3879
Author(s):  
Gayle M. Buller ◽  
Jolene A. Bradford ◽  
Jixiang Liu ◽  
William L. Godfrey
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Propidium Iodide ◽  
Cell Membranes ◽  
Fluorescent Dyes ◽  
Jurkat Cells ◽  
Dead Cell ◽  
Acetoxymethyl Ester ◽  
Alexa Fluor ◽  
Red Dye

Abstract With polychromatic flow cytometry becoming more prevalent, there is increasing interest in excluding dead cells from analyses without sacrificing the fluorophores already in use. We report several novel organic dyes that can identify stressed or dead cells in stained populations without compromising channels used for common fluorophores such as Alexa Fluor® 488, R-phycoerythrin (R-PE) and R-PE tandem dyes. Fixable violet and fixable aqua dead cell stains been developed that have peak emissions around 450 and 515 nm, respectively, and which can withstand aldehyde fixation, allowing their use with surface and intracellular labeling protocols. These amine reactive fluorescent dyes covalently label dead cells more brightly than live cells because the dye stains the cytoplasm of cells that have lost membrane integrity. (Figure 1A) These dyes stain equivalent dead cell populations versus ethidium monoazide bromide (EMA), but they do not require the additional photolysis step to cross-link EMA to the DNA of dead cells. SYTOX® red dead cell stain is a high-affinity nucleic acid stain that penetrates cells with damaged cell membranes, but will not cross uncompromised cell membranes. Cells stained with SYTOX red dye fluoresce bright red when excited with a red diode laser (Figure 1B), and can be used with fluorophores such as Alexa Fluor 488 dye and R-PE with little need for spectral correction. These properties, combined with a greater than 500-fold increase in fluorescence upon nucleic acid binding, make SYTOX red an optimal dead cell stain for use in flow cytometers equipped with red lasers. For measures of vitality, CellTrace™ calcein violet,AM dye is a metabolic probe that indicates intracellular esterase activity through the enzymatic conversion of the nonfluorescent, cell-permeant acetoxymethyl ester (AM) to a fluorescent violet-excited dye that is retained in the cell and emits fluorescence around 440 nm. Calcein violet,AM shows similar performance to calcein, AM, a common vitality reagent in flow cytometry and microscopy, and can be used in combination with impermeant DNA dyes such as SYTOX red dye or propidium iodide to identify live, injured and dead cells. (Figure 1C) For a violet-excited live/dead assay, the fixable aqua dead cell stain, with peak emission around 515 nm, can be combined with calcein violet,AM. Calcein violet,AM also can be used with Alexa Fluor 488 annexin V and propidium iodide to add a measure of enzymatic activity to the study of apoptosis. Together, these reagents provide multiple methods to add viability and vitality discrimination into standard immunostaining panels. Figure 1. Mixed live and heat-killed Jurkat cells stained with (A) fixable violet dead cell stain, (B) SYTOX red stain, and (C) a mixture of calcein violet,AM and SYTOX red dye. Figure 1. Mixed live and heat-killed Jurkat cells stained with (A) fixable violet dead cell stain, (B) SYTOX red stain, and (C) a mixture of calcein violet,AM and SYTOX red dye.

scholarly journals Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment

1998 ◽  
Vol 64 (7) ◽  
pp. 2697-2700 ◽  
Author(s):  
P. Lebaron ◽  
P. Catala ◽  
N. Parthuisot
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Salmonella Typhimurium ◽  
Viability Assessment ◽  
Bacterial Viability ◽  
Sytox Green ◽  
Nucleic Acid Stain

ABSTRACT The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations ofEscherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.

Development of a Mechanism-Based, DNA Staining Protocol Using SYTOX Orange Nucleic Acid Stain and DNA Fragment Sizing Flow Cytometry

2000 ◽  
Vol 286 (1) ◽  
pp. 138-148 ◽  
Author(s):  
Xiaomei Yan ◽  
Robert C. Habbersett ◽  
Julia M. Cordek ◽  
John P. Nolan ◽  
Thomas M. Yoshida ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Dna Staining ◽  
Staining Protocol ◽  
Dna Fragment ◽  
Nucleic Acid Stain

Highly chlorinatedEscherichia colicannot be stained by propidium iodide

2007 ◽  
Vol 53 (5) ◽  
pp. 664-670 ◽  
Author(s):  
M.-H. Phe ◽  
M. Dossot ◽  
H. Guilloteau ◽  
J.-C. Block
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Propidium Iodide ◽  
False Negative ◽  
Sodium Thiosulphate ◽  
Permeable Membrane ◽  
E Coli ◽  
Dna And Rna ◽  
Chlorine Bleach ◽  
Effect Of Chlorine

Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 μmol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.

Sign in / Sign up

Export Citation Format

Share Document