Propidium Iodide Staining Protocol

Propidium Iodide Staining Protocol – Annexin v and apoptosis, Immunophenotyping, Analysis of cell vitality and viability using calcein am / pi, Propidium iodide (pi) and hoechst 33342 double staining in cultured c6…, Neun and propidium iodide staining found no indications of pxn induced…, A) annexin v fitc propidium iodide (pi) binding assay for evaluation…

1 Environmental Toxicology Laboratory; National Institute of Chemical Physics and Biophysics; Akadeemia tee 23, 12618 Tallinn, Estonia 2 Department of Chemistry and Biotechnology; Tallinn University of Technology; Academia T15, 12618 Tallinn, Estonia

3 LEPABE – Laboratory for Process Engineering, Environment, Biotechnology and Energy; Department of Chemical Engineering; Faculty of Engineering; University of Porto; Rua Dr. Roberto Frias, 4200-465 Porto, Portugal

Propidium Iodide Staining Protocol

Combining membrane-impermeable DNA-binding propidium iodide (PI) spots with membrane-impermeable DNA-binding counterstance is a widely used approach to bacterial solubility spots. In this paper we show that PI staining of follower cells in biofilms can significantly underestimate the viability of bacteria due to the presence of extracellular nucleic acids. We show that gram-positive

In Vivo Propidium Iodide (pi) Staining Assessment In The Transplanted…

Can be cultivated after harvest. Confocal laser optical microscopy (CLSM) revealed that this false red cell layer is due to a double-stained cell subpopulation that has a green interior under the red layer indicating a spot on the outside of the intact membrane. Therefore, viability spot results in adaptive cells should always be validated by an alternative method for viability estimation, preferably by cultivation.

. PI can only cross membrane compromised bacteria and is therefore considered an indicator of membrane integrity. It attaches DNA and RNA inside dead cells or with reversible damaged membranes. For efficiency the PI stain is usually combined with a universal stain that crosses the intact membrane and stains DNA and RNA in all cells, allowing them to obtain the total number of cells. The most common example of such a co-spot is SYTO 9. While co-spot with PI and SYTO 9, SYTO 9 can enter all cells regardless of the integrity of the membranes, bind with DNA and RNA, and emit green fluorescence while will PI only insert cells with damaged membranes, DNA And connect with RNA and emit. a red fluorescent signal. With high attractiveness for DNA binding and enough for SYTO 9, PI replaces SYTO 9, when both spots come in contact with the same DNA causing a red fluorescent signal. As a result of the combination of these DNA-binding-based spots and membrane permeability, red signals from cells are considered “dead” and green signals “alive”.

. Although this principle is widely applied and proves to work well for a range of planktonic cultures, its limitations are uneven staining of SYTO 9 in competent and dead cells, incomplete replacement of SYTO 9 by PI, or energy during co-staining.

. It has also been shown that PI can in some cases give false dead signals entering solid cells with high membrane potential.

Propidium Iodide (pi) And Hoechst 33342 Double Staining In Cultured C6…

. The results of PI-based solubility spots are not always related to viability due to the solid but uncultivated condition (VBNC) of bacterial cells.

Another factor to consider when attaching cells to DNA / RNA-binding fluorophoes is that nucleic acids are not always localized just inside the bacterial cells and are surrounded by membranes. For example, extracellular DNA (EDNA) may be present in planktonic cultures at certain growth stages.

, And it also plays a major role in mature biofilms. The importance of eDNA in biofilm formation is demonstrated by the fact that DNase I prevents the formation of biofilm or isolated biofilm existing in some Gram-positive and Gram-negative bacterial species.

. The presence of DNA from non-solid sources (EDNA and DNA from dead cells) also presented the need for the use of study monoside (EMA), propidium monoside (PMA) or endonuclease (DNA I) by quantitative polymerase. Feedback (qPCR)

Propidium Iodide (pi) Staining Solution (50μg/ml) E Ck A161 Manufacturer

. All of the above treatment agents, EMA, PMA and DNase I intact membranes are impermeable DNA-targeting compounds targeting spatial DNA similar to PI depending on membrane integrity.

To get an overview of whether the presence of EDNA in biofilms is considered a factor that could interfere with PI-based fluorescent stains, we searched the Scopus database for “biofilm” and “iodide propidium” and we found “Extra 683 Results” when we add. Or the number of results in the “eDNA” research dropped to 43 indicating that when PI is used for biofilm spots, the potential presence of eDNA in this context is usually not taken into account. In the literature we find that PI is also used for eDNA staining

But there is no clear quantitative evidence that PI biofilm is not suitable for viability spots due to the presence of nucleic acid in the ECM biofilm. More surprisingly, membrane-based stain solubility intact waterproof DNA-binding stains, such as PI, are also used occasionally when studying EDNA.

. However, in some of the articles, signs of such threats can be found. For example, Giao and Kiwel saw some

Double Staining With Hoechst And Propidium Iodide (pi). The Images In…

Most of the older biofilms grown in tap water biofilm and rich media were red with PI and SYTO 9 co-spots, but being cultured with suspected red spots was not an indicator of dead cells but was caused by eDNA.

. From these sources it can be suspected that solubility spots based on PI in biofilm, although commonly used, can be severely affected by EDNA and underestimate solubility in biofilm. To address this possibility, we assessed the quantitative viability of follower cells using various spot and culture-based methods.

Epipholorescence microscopy (EM), flow cytometry (FCM) and confocal laser optical microscopy performed on iodine propidium (PI) and SYTO 9 stained adherent and harvesting bacterial cells were combined with camouflage. Stains of bacteria associated with PI may underestimate their effectiveness. Early (24 hours) Gram-negative biofilm

. Biofilms of these bacterial strains on the glass surface were made of saline buffer phosphate (PBS) to exclude the potential effects of osmotic strains on the bacterial membrane and possibly on stain solubility results.

Neun And Propidium Iodide Staining Found No Indications Of Pxn Induced…

After PI + SYTO 9 co-spots, the most coherent cells (96.35 ± 5.3%)

(Fig. 1a, 1b, 2a, 2b) while most (approximately 99%) planktonic cells from the respective upper biofilm suspensions are green with SYTO 9 on the filter (extra figure 1). This can generally be interpreted as showing differences in the different proportions of dead and living cells and the physiology of planktonic cells, showing better viability of planktonic cells. However, considering this difference it is difficult to explain that the follicle cells showed biofilm-specific aggregation in the microcolonies, no toxic agents were added, the samples were washed before staining and loose dead planktonic cells should be removed. Also, the proportion of red-spotted cells in early biofilms was surprisingly high. For example, using the same spot method, Wang

. Steam-hungry biofilms of PBS are most commonly used in oral health studies where most of the cells in the biofilms are green in color similar to zoo.

Propidium iodine (PI) and SYTO 9 (a, b), cut with fluorescein diacetate (FDA) (c, d) or by sonication, stained with PI and SYTO 9 and collected on filter (e, f). The pie chart shows the total number of cells on the surface and the proportion of stained signals PI, SYTO 9 and FDA marked in red, dark green and light green, respectively. The bar scale corresponds to 10 µm.

Conventional Apoptosis Assays Using Propidium Iodide Generate A Significant Number Of False Positives That Prevent Accurate Assessment Of Cell Death.

Figure 2. E. coli (a, c) and S. epidermidis (b, d) comparison of multiple approaches to evaluate the efficacy of follower cells in biofilms after harvesting by surface located (a, b) or ultrasonication (c, d). ) in the biofilm.

24-hour initial monolayer biofilm PBS attached to glass formed in situ (A, B) propidium iodide (PI) with SYTO9 or FDA and then microscope epipholorescence (EM) and signal calculation or harvesting (C, D) and cultivation for plates. Came. The calculations are co-attached with PI and SYTO 9 and analyzed by flow cytometry (FCM) or collected on a filter by EM and signal calculations. Results are presented as signal / cm

Where a signal is considered it corresponds to a single fluorescent cell or diplococcus (microscopy), CFU (culture) or FCM event. Ging live / dead in the FCM signal population was based on known levels of solid planktonic bacteria and killed ethanol. For an average and standard deviation of 4-6 independent values

10-16 independent values ​​for staining and filtering and plate counting with FCM are shown and only statistically significant differences (p 0.05; * <0.05; ** <0.01; *** <0.001 ; **** <0.0001)

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In biofilms, we also stained adherent cells with fluorescein disetate (FDA), which is not a DNA-binding, but a stain indicator of enzymatic activity that emits green fluorescent after enzymatic intracellular cleavage.

Total calculations (Figures 1c, 1d, 2a, 2b). Comparison of stain results on cells with FDA and PI + SYTO 9 shows that for both bacterial species, there is a statistically significant difference in FDA and SYTO 9 signal counts (Fig. 2a, 2b) but there is no significant difference between. FDA and PI or total (PI + SYTO 9) signal counts. Assuming that dead cells are not metabolically active, and starvation may even underestimate the number of solid cells based on FDA stains, this result sharply contradicts PI + SYTO 9 solubility stain results. These results show that most of the cells on glass surfaces are metabolically active with spots and PI while a minority of cells have potentially solid spots with SYTO 9..

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