Propidium Iodide (PI) or DAPI Staining of Unfixed Solid Tissues for Flow Cytometry

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4438
Author(s):  
Linda Rodgers
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dapi Staining ◽  
Solid Tissues

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

scholarly journals Chromosome doubling in Cattleya tigrina A. Rich

2019 ◽  
Vol 15 (11) ◽  
Author(s):  
Thays Saynara Alves Menezes-Sá ◽  
Maria de Fátima Arrigoni-Blank ◽  
Andréa Santos da Costa ◽  
Janay De Almeida Santos-Serejo ◽  
Arie Fitzgerald Blank ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Chromosome Doubling ◽  
Flow Cytometric Analysis ◽  
Leaf Tissue ◽  
Stomatal Index ◽  
Chromosome Duplication ◽  
Flow Cytometric ◽  
Young Leaves ◽  
Commercial Value

Chromosome doubling induction in orchids may benefit their production for resulting in flowers of higher commercial value, larger size and higher content of substances that intensify the color and fragrance when compared with diploid orchids. This work aimed to induce and confirm artificial polyploidization, using flow cytometry and stomatal analysis. Explants were treated with colchicine at concentrations of 0, 2.5, 7.5, and 12.5 mM, for 24 and 48 hours and with oryzalin, at concentrations of 0, 10, 30, and 50 μM, for three and six days. For the flow cytometric analysis, a sample of leaf tissue was removed from each plant, crushed to release the nuclei and stained with propidium iodide. In addition to flow cytometry, the ploidy of the antimitotic treated plants was evaluated by stomata analysis. Young leaves were used where the density, functionality and stomatal index were evaluated. Colchicine provided induction of satisfactory polyploidy in C. tigrina at all concentrations and times of exposure, obtaining a greater number of polyploid individuals in the concentration of 12.5 mM for 48 hours. Oryzalin did not induce chromosome duplication at the tested concentrations.

scholarly journals Use of Formalin-Fixed, Propidium Iodide-Stained Human Leukocytes as a Standard for Enumerating CD4+ T Lymphocytes in a Single-Platform Assay

2001 ◽  
Vol 8 (2) ◽  
pp. 397-401 ◽  
Author(s):  
G. M. Harrison ◽  
A. J. Bennett ◽  
M. Moody ◽  
G. F. Read ◽  
P. E. Williams
Keyword(s):  
Flow Cytometry ◽  
Blood Cell ◽  
Propidium Iodide ◽  
Internal Standard ◽  
Inexpensive Method ◽  
Normal Cells ◽  
Blood Samples ◽  
Cell Enumeration ◽  
Whole Blood Samples ◽  
Formalin Fixed

ABSTRACT A new, inexpensive method is described that enables lymphocytes to be enumerated very precisely. Normal leukocytes were simultaneously stained and fixed with a propidium iodide-paraformaldehyde solution. The preparation obtained (CellBeads) was used as an internal standard for cell enumeration by flow cytometry and was stable at 4°C for at least 60 days. Unlike synthetic beads, the CellBeads behaved similarly to normal cells during red blood cell lysis and cell washing procedures. When known numbers of CellBeads were added to whole-blood samples and the numbers of CellBeads and lymphocytes were determined, highly reproducible and accurate enumerations were obtained—far more so than when synthetic beads were used. This inexpensive method is suitable for routine use.

Pannexin channels increase propidium iodide permeability in frozen–thawed dog spermatozoa

2017 ◽  
Vol 29 (11) ◽  
pp. 2269 ◽  
Author(s):  
J. L. Torres ◽  
J. Palomino ◽  
R. D. Moreno ◽  
M. De los Reyes
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Analysis Of Variance ◽  
Propidium Iodide ◽  
Membrane Channels ◽  
Flow Cytometry Data ◽  
Viable Cells ◽  
Head Segment ◽  
Relationship Of ◽  
The Relationship

Pannexins (Panx) are proteins that form functional single membrane channels, but they have not yet been described in dogs. The aim of the present study was to detect Panx1, Panx2 and Panx3 in frozen–thawed dog spermatozoa using flow cytometry and immunofluorescence analyses, evaluating the relationship of these proteins with propidium iodide (PI) in frozen–thawed spermatozoa. Fresh and frozen–thawed dog spermatozoa from eight dogs were preincubated with 3 μM PI with or without 15 μM carbenoxolone (CBX) or 1 mM probenecid (PBD), two Panx channel inhibitors, and then incubated with rabbit anti-Panx1, anti-Panx2 and anti-Panx3 antibodies (1 : 200). Panx immunolocalisation was assessed by fluorescence microscopy. Flow cytometry data were evaluated by analysis of variance. All three Panx proteins were found in dog spermatozoa: Panx1 was mostly localised to the acrosomal and equatorial segment, Panx2 was found in the posterior region of the head and tail and Panx3 was localised to the equatorial and posterior head segment. The percentage of PI-positive cells determined by flow cytometry was reduced (P < 0.05) in the presence of Panx inhibitors. These results show that Panx proteins are present in dog spermatozoa and increase PI permeability in frozen–thawed dog sperm, suggesting that the percentage of PI-positive spermatozoa used as an indicator of non-viable cells may lead to overestimation of non-viable cells.

The genome sizes of Hordeum species show considerable variation

Genome ◽  
1996 ◽  
Vol 39 (4) ◽  
pp. 730-735 ◽  
Author(s):  
Juha Kankanpää ◽  
Alan H. Schulman ◽  
Leena Mannonen
Keyword(s):  
Flow Cytometry ◽  
Hordeum Vulgare ◽  
Genome Size ◽  
Nuclear Dna ◽  
Size Variation ◽  
Nuclear Dna Content ◽  
Meiotic Pairing ◽  
Genome Size Variation ◽  
Dapi Staining ◽  
Hordeum Vulgare Ssp

Hordeum, distributed worldwide in temperate zones, is the second largest genus in the tribe Triticeae and includes diploid, tetraploid, and hexaploid species. We determined, by DAPI staining and flow cytometry, the nuclear DNA content for 35 accessions of the genus Hordeum, from a total of 19 species, including specimens of 2 cultivars and 2 landraces of Hordeum vulgare ssp. vulgare as well as samples of 12 Hordeum vulgare ssp. spontaneum populations. Genome sizes ranged from 5.69 to 9.41 pg for the G1 nuclei of the diploids, and from 13.13 to 18.36 pg for those of the tetraploids. This constitutes a 1.7-fold variation for the diploids, contrasting with a 4% variation previously reported. For H. vulgare ssp. vulgare (barley), the accessions examined differed by 18%. These variations in genome size cannot be correlated with meiotic pairing groups (I, H, X, Y) or with proposed phylogenetic relationships within the genus. Genome size variation between barley accessions cannot be related to status as cultivated or wild, or to climatic or geological gradients. We suggest these data may indicate rapid but sporadic changes in genome size within the genus. Key words : barley, Hordeum, Triticeae, genome size, flow cytometry.

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.

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