Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry

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.

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Genome Size Determination Using Flow Cytometry of Propidium Iodide-Stained Nuclei

Methods in Molecular Biology - Molecular Methods for Evolutionary Genetics ◽

10.1007/978-1-61779-228-1_1 ◽

2011 ◽

pp. 3-12 ◽

Cited By ~ 42

Author(s):

Emily E. Hare ◽

J. Spencer Johnston

Keyword(s):

Flow Cytometry ◽

Genome Size ◽

Propidium Iodide ◽

Size Determination

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Data-Driven Compensation for Flow Cytometry of Solid Tissues

Advances in Bioinformatics ◽

10.1155/2011/184731 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11 ◽

Cited By ~ 3

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.

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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.2.397-401.2001 ◽

2001 ◽

Vol 8 (2) ◽

pp. 397-401 ◽

Cited By ~ 5

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.

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

Reproduction Fertility and Development ◽

10.1071/rd16267 ◽

2017 ◽

Vol 29 (11) ◽

pp. 2269 ◽

Cited By ~ 1

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.

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