Blue Strip of Dna Came From What Cell

Propidium Iodide

PI fluoresces red when bound to nucleic acids, whereas AO fluoresces green when bound to double-stranded DNA and red when bound to singlestranded DNA or RNA.

From: Cellular Transplantation , 2007

Tumor Immunology and Immunotherapy – Molecular Methods

Leepakshi Khurana , ... Georgios Pantouris , in Methods in Enzymology, 2019

5 Cell-cycle arrest analysis

In contrast to healthy cells, cancer cells develop an increased capacity to proliferate. To ensure constant proliferation, cancer cells tend to hijack the normal cell-cycle machinery and alter the checkpoints at the G₁/S or G₂/M phase. These checkpoints are the surveillance mechanisms by which healthy cells are regulated to follow normal growth and differentiation and thus alterations in the cell cycle are one of the major mechanisms by which cancer propagates. Changes in cell-cycle upon treatment with inhibitors of immunomodulatory proteins allow a simple readout for screening compounds with potential anti-cancer activity. For example, IDO1 can cause cell-cycle arrest of T cells infiltrating the tumor microenvironment, rendering the immune system inactive against tumor cells (Labadie, Bao, & Luke, 2019). Thus, targeting of immunomodulatory molecules like IDO1 may alter cell-cycle checkpoints, which can be studied using flow cytometry.

Many univariate and multivariate flow-cytometric methods are available to quantify the percentage of cells in different phases of the cell cycle. A small molecule with potential anti-cancer activity can cause changes in the cell-cycle dynamics, which can result in arresting cancer cells at one of the checkpoints. The simplest method of cell-cycle analysis involves treatment of cells with propidium iodide, which binds to DNA by intercalating between bases and allows quantification of cellular DNA content in different phases. These measurements can be used to segregate cells in the three cell-cycle phases, i.e., G ₀/G₁ phase, S phase and G₂/M phase. The gating strategy is based on the ploidy of cells, which depends upon the cell-cycle phases. While cells in G₀/G₁ phase have 2n ploidy, the cells found in G₂/M phase have 4n ploidy and thus double the amount of DNA content. Therefore, cells in the G₂/M phase have double the fluorescence intensity than those in the G₀/G₁ phase and cells in these two phases are seen as two, separated peaks. The cell population between the two peaks represents cells in the S phase. The procedure for cell-cycle analysis using propidium iodide is outlined below. As an example, we use HL-60 cells, an acute promyelocytic leukemia cell line, in the absence or presence of a small molecule modulator.

5.1 Equipment, reagents and software

Propidium iodide 1 mg/mL (Sigma Aldrich, P4864)

RNAse (Qiagen, 19101)

Water bath

Triton X-100 (Sigma Aldrich, T8787)

HL-60 cells (American Type Culture Collection (ATCC), CCL-240)

RPMI 1640 with l-Glutamine (Thermo Fisher Scientific, 11875093)

Fetal bovine serum (FBS) (Life Technologies, 16000-044)

Penicillin, streptomycin (Fisher Scientific Company LLC, 15140122)

PBS 10 × (Thermo Fisher Scientific, 10010023)

Ethanol (Fisher Scientific, 22-032-600)

Falcon tubes conical centrifuge tubes (15 mL) (Corning, 352196)

T-75 cell culture flasks (Thermo Fisher Scientific, 156499)

Centrifuge (compatible with 15 mL falcon tubes)

LSRII flow cytometer (Becton Dickinson)

Test tube with cell strainer snap cap (Corning, 352235)

FlowJo software (for plotting data and analysis)

5.2 Steps and annotations

5.2.1 Cell culture

1.

Culture HL-60 cells in a T-75 flask containing RPMI-1640 medium with 2 mmol/L glutamine, 10% v/v FBS and 1% v/v penicillin/streptomycin. Incubate the cells at 37 °C and 5% CO₂ and maintain the cell density between 1 × 10⁵ and 1 × 10⁶ viable cells/mL. Change the medium every 2 to 3 days.

2.

Bring the cells to passage 3 before setting up any experiments and use them up to passage 8.

3.

For assessing the impact of a small molecule modulator on cell cycle, starve HL-60 cells by resuspending them in RPMI-1640 medium with 2 mmol/L glutamine, 0.5% FBS and 1% v/v penicillin/streptomycin. Starvation synchronizes the cells. Plate these cells in a six-well plate, at a density of 1 × 10⁶ cells/well. Incubate overnight at 37 °C and 5% CO₂.

4.

On day 2, treat the cells with DMSO (0.1% v/v final) or different concentrations of a small molecule modulator dissolved in DMSO. Incubate the cells at 37 °C and 5% CO₂, for 24 h.

Note: Initial trials for cell-cycle analysis may include drug treatments for 12–48 h to optimize the time of incubation with the test compound.

5.

After 24 h, harvest the cells for propidium iodide (PI) staining.

5.2.2 PI staining solution

Following is the recipe for preparing the PI staining solution:

PI (0.02 mg/mL)

Triton X-100 (0.1% v/v)

RNAse A (0.2 mg/mL)

PBS

The PI staining solution should be prepared fresh every time. Each sample is treated with 1 mL of PI staining solution. The solution is prepared in PBS and kept on ice throughout the staining process

Note: Treatment of cell samples with RNAse A is necessary since PI binds to both RNA and DNA

Triton X-100 treatment ensures complete permeabilization of cells so that the dye can freely access DNA

5.2.3 Fixation and staining with PI staining solution

1.

Harvest approximately 1 × 10⁶ HL-60 cells (treated with DMSO or different concentrations of a small molecule modulator) into 15 mL falcon centrifuge tubes. To remove medium and pellet the cells, centrifuge the tubes at 500 × g for 5 min. Carefully, remove the supernatant. Keep the cells on ice.

2.

To wash off the remaining medium, resuspend the cells in 5 mL of ice-cold PBS. Centrifuge the tubes at 500 × g for 5 min to pellet the cells.

3.

Resuspend the cells in 0.5 mL PBS by pipetting up and down. Using a glass pasteur pipette, transfer the cell solution, drop-wise, into 4.5 mL of 70% ethanol for fixation. Keep vortexing the ethanol solution gently while adding the cell solution. This ensures minimal cell clumping which is necessary for DNA content analysis. Allow the cells to be fixed in ethanol for at least 2 h at 4 °C.

Note: The choice of fixative for cell-cycle analysis is critical. Ethanol is the preferred fixative for cell-cycle analysis as it is a dehydrating fixative along with permeabilizer. Thus, the dye can easily access DNA and better quality profiles (low coefficient of variation (CV)) are gained. However, if simultaneous detection of any surface markers is needed, aldehydes (cross-linking fixatives) such as paraformaldehyde should be used for fixation since ethanol fixation is often incompatible with many surface markers. The drawback of using aldehydes for fixation is that it leads to poorer quality profiles (high CV). Also, paraformaldehyde does not permeabilize cells, thus necessitating strong permeabilizing agents to be used for allowing the dye to access DNA. After ethanol fixation, the cells may be stored for a week before analysis. However, the quality of data is better when the samples are fresh.

4.

Centrifuge the cells at 500 × g for 5 min. The pellet is very flaky at this point and should be handled carefully. Discard the supernatant. Wash the cells with 5 mL of PBS to ensure compete removal of ethanol before treating with PI staining solution. Centrifuge the cells again and remove PBS.

5.

Resuspend the cells in 1 mL of PI staining solution (see recipe above) and let it sit at room temperature for 20 min, in the dark.

6.

After 20 min, filter the cells into tubes with filter tops. Filtering cells help to avoid cell-clumping and clogging the nozzle of the flow-cytometry instrument.

Note: It is not necessary to wash the cells to remove the PI staining solution before filtering the cells.

5.2.4 Using flow cytometry instrument and data analysis

1.

Adjust the flow cytometry instrument to read PI fluorescence. The absorption maxima for PI fluorescence is 536 nm and a blue laser (488 nm) is optimal for excitation of PI. Ensure that the recordings are done for area, height and width of the signal and the recordings are done on linear scale, unlike most of the flow-cytometric analyses, which are done on logarithmic scales. Set a low flow rate (< 400 events/s) for optimal fluorescence resolution.

2.

Exclude cell debris by creating a gate on the FSC versus SSC plots and exclude cells with very low forward and side scatter (Fig. 4A ).

3.

The cells within the gate are further analyzed to remove doublets. To remove doublets, plot the pulse height or width versus pulse area and exclude the cells with higher width or disproportional area (Fig. 4B).

Note: Doublet discrimination is critical for proper cell-cycle analysis. Doublets of G₀/G₁ cells demonstrate signal intensities similar to singlets of G₂/M phase, which can skew the overall analysis.

4.

The singlet, live cells are further analyzed for PI fluorescence. Create a histogram of PI fluorescence versus number of events. Since the DNA content of cells in the G₂/M phase is double the DNA content of G₀/G₁, the histogram will show two clear peaks (Fig. 4C). The cell population between the two peaks represent the S phase.

5.

Apply the Watson-Pragmatic model in the FlowJo software to the singlet, live cells. This will give the % cells in each phase. Constraints can be applied on the position of the G₂ peak (~ 1.95 × G₁) and the CV of G₂ peak (G₁ CV) to improve the overall CV of the cell-cycle profile.

Note: Ideally, the position of the G₂ peak should be at 2 × G₁ peak. However, due to peak broadening effects, the peak is slightly shifted, which is why the number to be added in the constraints is not 2 × G₁ for the position of G₂ peak.

6.

To evaluate the impact of a small molecule on cell cycle and to demonstrate arrest in any phase, overlay the histograms of DMSO (control) and compound treatments (Fig. 4D). Compare the % cells in each phase for the different concentrations of test compound to control. Increase in the % cells in any phase of cycle demonstrates arrest.

5.2.5 Using markers for different phases of cell-cycle

PI staining allows identification of the three distinct phases of cell cycle and is a good starting assay for identification of compounds with potential anti-cancer activity. However, some prediction models such as Watson Pragmatic or Dean-Jett-Fox need to be applied in the software (e.g., FlowJo) to get % cells in each phase since S-phase cells have slight overlaps in both G₀/G₁ phase and G₂/M phase. Also, PI staining cannot be used to distinguish between G₀ and G₁ cells or G₂ and M cells. Further elucidation of the detailed cell-cycle status can be done by including staining markers, which are specific for different phases of cell cycle. For example, concurrent staining of cells with Pyronin Y, which stains RNA and Hoechst 33342, which stains DNA, allows segregation of cells in the G₀ phase (lower RNA levels are seen in G₀ than G₁ while levels of DNA are similar in both phases) from G₁ cells (Fig. 4E). Some of these cell-cycle specific markers are shown in Table 5 along with suggested DNA dyes for multivariate analysis.

Table 5. Markers that can be used with DNA dyes for multivariate analysis of cell-cycle phases.

Phase of cell-cycle to be analyzed	Antibody/stain	Additional comments
M phase	Anti-Phospho-histone H3	Double staining with PI allows separation of G₂ and M phase cells. M phase cells stain high with phospho-histone H3 (Shen, Vignali, & Wang, 2017)
G₀ phase	Pyronin Y	Double staining with Hoechst 33342 allows separation of G₀ cells from G₁, S and G2/M. Dye binds to RNA, which is low in G₀ cells compared to cells in other phases (Schinke et al., 2015)
S phase	Anti-BrDU	Double staining with PI (or other DNA dyes) allows separation of S phase which stains high for BrDU versus G₀/G₁ and G₂/M phase with low BrDU staining (Mikes et al., 2014)
G₀ phase	Anti-Ki67	Ki67 is a nuclear proliferation antigen, which stains G₁, S, G₂ and M phase but does not stain G₀ cells and allows separation of G₀ cells from other phases. Can be used in multivariate analysis with PI or other DNA dyes (Gerdes et al., 1984)
G₂ phase	Anti-Cyclin A	Cyclin A staining allows differentiation of G₂ cells from M phase cells as G₂ cells show high levels of cyclin A staining, which drops during M phase. Combined with DNA dyes, it allows differentiation of the four phases and subpopulations of cells (Darzynkiewicz, Gong, Juan, Ardelt, & Traganos, 1996)
S phase	Anti-PCNA	Proliferating cell nuclear antigen (PCNA) stains proliferating cells in the S phase. Combined with DNA dyes, this marker can be used to segregate S phase cells from G₀/G₁ cells from G₂/M cells (Larsen, Landberg, & Roos, 2001)

The markers used, the phase of cells that are stained by these markers and additional comments about the DNA dyes are provided.

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Primary Cultures of Cardiac Myocytes as In Vitro Models for Pharmacological and Toxicological Assessments

ENRIQUE CHACON , ... JOHN J. LEMASTERS , in In Vitro Methods in Pharmaceutical Research, 1997

1 Propidium iodide dye exclusion assay

The propidium iodide dye exclusion assay is performed in a Krebs–Ringer–HEPES buffer (KRH: 115 mM NaCl, 5 mM KCl, 1 mM KH ₂PO₄, 1.2 mM MgSO₄, 2 mM CaCl₂, and 25 mM HEPES at pH 7.4) containing 30 μM propidium iodide. In the absence of cells, propidium iodide exhibits an excitation maximum near 500 nm and an emission maximum near 625 nm. Binding of propidium iodide to DNA causes a red shift of the excitation maximum to 540 nm and the emission maximum to 640 nm, with a two- to threefold increase in fluorescence intensity. A multi-well fluorescence scanner equipped with appropriate excitation and emission filters can be used to record fluorescence from 24- or 96-well microtiter plates. For cytotoxicity screening, myocytes are cultured in 96-well microtiter plates (50 000 cells per well) and treated with varying concentrations of test agents. Typically, we scan plates every 15 or 30 min. Between measurements, plates are kept in a 37°C air incubator. Increasing fluorescence signifies loss of cell membrane integrity. Maximal change of fluorescence corresponds to 100% cell death. At the end of the experiment, 25 μM digitonin or 5 μM Triton X100 is added to each well to permeabilize all cells and label all nuclei with propidium iodide. Fluorescence is measured again to determine a value corresponding to 100% cell death. Percentage viability (V) is calculated as V = 100 (X–A)/(B–A) where A is initial fluorescence, B is fluorescence after addition of digitonin or Triton X100, and X is fluorescence at any given time. The propidium iodide asssay allows continuous measurements of cell viability over time. However, as in any fluorescence assay, appropriate controls must be performed to assure that test agents are not themselves fluorescent or cause any other fluorescence interference with the fluorescent marker.

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Cell Biological Applications of Confocal Microscopy

David L. Gard , in Methods in Cell Biology, 2002

G Nuclear Stains

+ Propidium Iodide (intercalating DNA dye). Commercially available from Molecular Probes (Eugene, OR; #P-1304). Works best with methanol-fixed stage 0 oocytes or maturing eggs. Counterstain with fluorescein-conjugated secondary antibodies.

+ Ethidium homodimer (intercalating DNA dye). Commercially available from Molecular Probes (Eugene, OR; #E-1169). Works best with methanol-fixed stage 0 oocytes or maturing eggs. For dual fluorescence applications, counterstain with fluorescein-conjugated secondary antibodies.

Note: PI and EH also stain cytoplasmic dsRNA, which can be reduced by pretreating samples with RNAse.

+++ YO-PRO-1 (cyanine DNA dye). Commercially available from Molecular Probes (Eugene, OR; #Y-3603). Works best with methanol-fixed stage 0 oocytes, eggs, and embryos. Some reduction in staining of aldehyde-fixed oocytes. For dual fluorescence applications, counterstain with Texas-Red-conjugated secondary antibodies.

++ BO-PRO-3 (cyanine DNA dye). Commercially available from Molecular Probes (Eugene, OR; #B-3586). Works best with methanol-fixed stage 0 oocytes, eggs, and embryos. Some reduction in staining of aldehyde-fixed oocytes. Worked best for single labeling.

Acknowledgments

Over the years, a number of individuals have contributed to the observations discussed in this chapter, including M. Schroeder, A. Roeder, B. Error, A. Friend, D. Affleck, P. Jenkins, B.-J. Cha, B. Becker, and S. Romney, and the author thanks them for their contributions. Special thanks are due to Dr. Edward King for his invaluable assistance with the confocal microscope facility. The work described has been supported by grants from the National Institute of General Medical Studies, National Science Foundation, and University of Utah Research Committee.

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Monitoring vesicular trafficking in cellular responses to stress - Part A

D. Liu , ... C. Brenner , in Methods in Cell Biology, 2021

2.2.1 Materials, reagents and equipment

(1): Propidium Iodide (PI, Invitrogen, P3566), 1 mM stock solution in PBS, pH 7.4, (Gibco™ 10010031)
(2): DMEM (Dulbecco's Modified Eagle Medium), high glucose, GlutaMAX™ Supplement, (Gibco™, 10566016)
(3): Lysis buffer: 25 mM Tris, HCl (pH 7.6) 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS
(4): 96 Well-TC-treated microplates (flat bottom) (Corning®, CLS3596)
(5): Tecan Infinite 200 spectrofluorimeter (Männedorf, Switzerland) or similar spectrofluorimeter equipped with a microplate reader to record fluorescence at λex: 530 nm and λem: 620 nm

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Nanomedicine

Yu Pan , ... Willi Jahnen-Dechent , in Methods in Enzymology, 2012

4.3.1 Protocol III

4.3.1.1 Determination of apoptosis versus necrosis

Annexin V and PI are double-staining probes for apoptosis by detecting the externalization of phosphatidylserine and membrane integrity. The apoptotic cells externalize their phosphatidylserine early in apoptosis when the cell membrane is still intact. Therefore, the early apoptotic cells have a positive annexin V, but a negative PI signal. In contrast to apoptotic cells, necrotic cells lose membrane integrity and both annexin V and PI will penetrate the leaky cell membrane to stain intracellular phosphatidylserine (annexin V) and nuclear DNA (PI). The test cannot discriminate between apoptotic cells at the late stage (secondary necrosis) and necrotic cells. Thus, time course measurements are required to determine the cell death pathway.

(1): Cells are seeded into 6-well plates (HeLa 40,000 cells/well) and incubated for 72 h at 37 °C with 5% CO₂ prior to the addition of nanoparticles. The seeding density is adjusted depending on the growth rate of the cell lines used and on the incubation duration. At least 20,000 cells are required at the end of the experiment to allow cell analysis by flow cytometry in the untreated samples.
(2): After 72 h of incubation, nanoparticles at the desired concentrations are applied.
(3): After the exposure to the nanoparticles, the supernatant containing detached apoptotic or necrotic cells together with the trypsinized cells are collected and washed twice with binding buffer followed with the addition of fluorescein isothiocyanate (FITC)-labeled annexin V.
(4): Annexin V binding to phosphatidylserine is calcium dependent. Therefore, the binding buffer must contain 2.5 mM calcium chloride throughout all steps including washing.
(5): The cells are incubated at room temperature for 15 min. Thereafter, an aliquot of the PI stock solution (10 μl, 50 μg/ml) is added to each well, and cells are further incubated for 5 min before one final wash in binding buffer. For each experiment, untreated cells serve as a negative control and cells incubated for 24 h with staurosporine (0.2 μM) serve as a positive control for apoptosis. Twenty thousand cells are counted for each sample by flow cytometry. Results are analyzed by CELLQuest software (Becton-Dickinson) (Fig. 11.3).

Figure 11.3. Flow cytometric determination of living, apoptotic, and necrotic HeLa cells treated with test compounds. Cells are scored for annexin V/PI double staining to estimate the relative amounts of live cells (annexin V/PI double negative, bottom left quadrant), apoptotic cells (annexin V-positive/PI-negative, bottom right quadrant), and necrotic cells (annexin V/PI-double positive, top right quadrant), respectively. The percentages of cells are given for each quadrant. (A) HeLa untreated, (B) HeLa 0.2 μM staurosporine, 24 h, (C) HeLa 110 μM Au1.4MS, 48 h.

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Apoptosis

Po Chen , John M. Abrams , in Methods in Enzymology, 2000

Reagents

PI solution: Propidium iodide (50 μg/ml), 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100

1.

Plate exponentially growing cells at 10⁶/ml in six-well plates or 35-mm plates, 2 ml/well, and incubate for several hours to overnight.

2.

Treat as desired.

3.

Gently wash the cells off the plates, save 1 ml for protein gel if needed, and put 1 ml into an Eppendorf tube.

4.

Spin down cells at 5000 rpm for 5 min at room temperature.

5.

Aspirate the supernatant and resuspend the cells with 1 ml of 2% (v/v) Formaldehyde in PBS; fix for 15 min.

6.

Spin at 5000 rpm for 5 min; resuspend the cell pellet in 0.5 ml of PI solution.

7.

Keep the cells at 4° overnight in the dark, and analyze within 24 hr.

[Note: We typically analyze samples on a Becton Dickinson (San Jose, CA) FACScan flow cytometer using LYSIS II software. The settings we use for the FACScan are as follows: FSC, linear E.00; Amplifier, 9.99; SSC, log 260; FL1, OFF; FL2, OFF; FL3, log 334. All resolutions are set at 1024.]

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Mesoporous Silica-based Nanomaterials and Biomedical Applications, Part B

Michihiro Nakamura , in The Enzymes, 2018

4.1.1 Macroscopic and Multimodal Imaging of Gastrointestinal Tract

Thiol-organosilica nanoparticles containing PI for multicolor fluorescence and X-ray computed tomography were applied for dual modal noninvasive functional gastrointestinal tract imaging [70]. Orally administered PI particles were observed in the gastrointestinal tract (GIT) using fluorescent imaging devices and X-ray CT. An in vivo fluorescence imaging system could detect the near-infrared fluorescence of PI particles in the GIT specifically. In addition, multipurpose zoom fluorescence microscopy was used to noninvasively visualize the real-time passage of particles and the movement of the GIT. The passage and distribution of particles over time in the GIT were demonstrated using X-ray CT. A correlation analysis between the fluorescent and X-ray CT data demonstrated the characteristics, limitations, and novel potential for noninvasive functional GIT dual modal imaging (Fig. 9).

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Advances in Cellular Neurobiology

H.G.J.M. Kuypers , A.M. Huisman , in Advances in Cellular Neurobiology, 1984

II Development of the Multiple Retrograde Fluorescent Tracer Technique for Demonstrating Axon Collaterals

The first steps toward the development of multiple fluorescent tracer techniques were made by Kristensson (1970) and by Steward and Scoville (1976), who demonstrated that Evans blue (EB) combined with bovine albumin (BA) is transported retrogradely through axons to their parent cell bodies in the same way as HRP. EB can be demonstrated in these cell bodies by means of fluorescence microscopy; in formalin-fixed material, the EB-labeled cell bodies show a flaming red fluorescence when illuminated with light of 550-nm wavelength (Kristensson, 1970; Kristensson et al., 1971; Steward and Scoville, 1976). Therefore, the retrograde neuronal labeling by means of Evans blue was studied in greater detail (Kuypers et al., 1977). The findings showed that EB without bovine albumin may give retrograde labeling of neuronal cell bodies equal to or even better than EB combined with BA. Thus, when injections of EB (10% in water) in the tongue or the caudate putamen in rat were followed by 1 or more days survival, the hypoglossal neurons, in the one case, and the neurons in the center-median and the substantia nigra pars compacta (SNC), in the other, showed flaming red fluorescence. This red fluorescence was studied in frozen sections which were cut from formalin-fixed material, mounted on slides, and air dried, with no coverslip. They were studied with the aid of a Leitz Ploemopack fluorescence microscope equipped with a high-pressure lamp of 100 W and with filter-mirror systems A, D, and N2, which provide excitation light wavelengths of approximately 360, 390, and 550 nm. The flaming red fluorescence of the EB-labeled neurons could be clearly observed with filter-mirror system N2 (550-nm wavelength), but only at a magnification of 25× or higher. However, lightly EB-labeled neurons were diffiuclt to differentiate from nonlabeled neurons, which at 550 nm show a red, granular autofluorescence.

The efficacy of the combination of EB with HRP for double retrograde labeling of neurons was tested by injecting these substances together in caudate putamen of rats, followed by a 1-day survival time. In these cases, many double-labeled neurons were present in the substantia nigra. These neurons must have been double labeled through single axons. When viewed with filter-mirror system A (360-nm excitation wavelength), the neurons displayed HRP granules in the cytoplasm, but when viewed with filter-mirror system N2, they showed red fluorescence only of the nucleus. The absence of the red fluorescence in the cytoplasm was probably due to the oxidation of EB by H₂O₂ in the presence of HRP, which accumulates in the cytoplasm, but does not enter the nucleus. However, since the red fluorescence of the nucleus in the EB–HRP double-labeled neurons was sometimes difficult to detect, the combination of EB and HRP was regarded as less than satisfactory for retrograde double labeling.

In a new series of experiments, an attempt was made to find another tracer which could be combined with EB in double-labeling experiments. For this purpose, the retrograde transport of a large series of substances was tested (Kuypers et al., 1977) using the rat nigrostriatal system as a model. All of these tracers were transported retrogradely after injections in termination areas of fiber systems as well as after injections in the fiber bundles. In the fluorescence microscope these tracers can be observed in the parent cell body only at higher magnification (i.e., with an objective lens of 25× or higher). Thus, the retrograde transport of diamidinophenylindole (DAPI) (2.5% in water) and primulin (10% in water) was discovered.

Neurons retrogradely labeled with DAPI display a bright blue fluorescence of the nucleus, including the nucleolus, and a duller blue fluorescence of the cytoplasm when viewed with filter-mirror system A. Moreover, the labeled neurons are frequently surrounded by blue fluorescent glial nuclei. When viewed with filter-mirror system D (390-nm excitation wavelength), DAPI-labeled neurons show green fluorescence.

Primulin-labeled neurons, when viewed with filter-mirror systems A and D, display golden fluorescent granules in the cytoplasm, but exhibit no labeling of the nucleus. The primulin-fluorescent granules can be easily differentiated from the autofluorescent clumps which are also located in the cytoplasm, but which fluoresce brown-yellow at 360 nm. However, in neurons in which the cytoplasm is filled with these autofluorescent clumps, the primulin granules may be difficult to detect. A similar labeling is obtained with the retrograde fluorescent tracer 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), which, in contrast to the vast majority of the other tracers, seems to be taken up only by terminals and not by broken axons (Schmued and Swanson, 1982). The combination primulin and DAPI (DAPI/Pr) has the advantage that both the nucleus (with DAPI) and the cytoplasm (with DAPI and Pr) are labeled. When EB and DAPI/Pr were injected together in the rat caudate putamen, many double-labeled neurons were present in the center-median parafascicular complex and SNC. These neurons, which must have been double labeled through single axons, displayed a flaming red EB fluorescence when viewed with filter-mirror system N2, and a blue and golden granular fluorescence when viewed with filter-mirror system A (Figs. 1 and 2).

In view of these findings, the combination of EB and DAPI/Pr seemed suitable for use in double-labeling experiments aimed at demonstrating the existence of divergent axon collaterals. In order to test this, the fiber projections from the mammillary bodies to the thalamus and mesencephalon were used as a model (Van der Kooy et al., 1978) because, according to Cajal (1952), they are at least in part established by divergent axon collaterals. EB was injected in rat anterior thalamus and DAPI/Pr in rat mesencephalic midline. After a survival time of 4 days, many EB–DAPI/Pr double-labeled neurons were present in the lateral mammillary nucleus and in the medial portion of the medial nucleus. When EB and DAPI/Pr were injected in the left and the right anterior thalamus, respectively, many double-labeled neurons were present in the lateral mammillary nucleus on both sides.

These findings clearly showed that EB and DAPI/Pr could be used as retrograde tracers in double-labeling experiments. However, in double-labeled neurons, the EB fluorescence and the DAPI/Pr fluorescence are less pronounced than in single-labeled neurons. EB and DAPI/Pr were also used in experiments aimed at studying the existence of axonal branching in the ascending raphe and nigral projections (Van der Kooy and Kuypers, 1979), and in experiments aimed at clarifying the existence of axonal branching in the nigrafugal connections (Bentivoglio et al., 1979a). In the latter study, it was demonstrated that the projections from the pars reticulata of the substantia nigra to the tectum and the thalamus are at least in part established by divergent axon collaterals of the same neurons, as has been confirmed in electrophysiological experiments (Niijma and Yoshida, 1982). Further, in this study (Bentivoglio et al., 1979a), the efficacy of EB and DAPI/Pr in retrograde labeling of pars reticulata neurons from the thalamus and the superior colliculus was found to be comparable to that obtained with HRP, as demonstrated by the DAB technique (Graham and Karnovsky, 1966; Mesulam et al., 1982).

In several largely unpublished experiments, long-distance transport of DAPI/Pr was found to be somewhat inconsistent, especially in cat; in some cases, beautiful labeling of cortical and brain stem neurons was obtained from the spinal cord, whereas in other cases no such labeling occurred. Therefore, the search for other fluorescent retrograde tracers was continued. It was then found that bisbenzimide (Bb) and propidium iodide (PI) are transported retrogradely over long distances, e.g., from the thoracic spinal cord to the sensorimotor cortex in rat and cat ( Kuypers et al., 1979). These two substances, however, produce an entirely different type of retrograde neuronal labeling.

Nigral neurons, which are retrogradely labeled after injections of bisbenzimide (10% in water) in caudate putamen, display a yellow-green granular fluorescence in the neuronal nucleus, which also shows a pronounced yellow-green fluorescence of its membrane and a pronounced yellow-green fluorescent ring around its nucleolus. In heavily labeled neurons, bright yellow fluorescent granules are also present in the cytoplasm. This type of fluorescence is obtained when viewing the neurons with filter-mirror system D, but when using filter-mirror system A, the nucleus shows a bluish-green instead of yellow-green fluorescence (Fig. 2). In these experiments, with long survival times relative to the transport distance (e.g., several days for transport from striatum to substantia nigra in rats), many fluorescent glial nuclei were also present around the retrogradely labeled neurons (Fig. 2). These glial nuclei can be easily distinguished from the neuronal nuclei because the latter characteristically show a fluorescent ring around the nucleolus. The labeling of the glial nuclei was thought to be due to Bb migration from the neuronal cell body into the glial cells associated with these neurons; because of its affinity to nucleotides, the Bb primarily labeled the nuclei of these cells. It was not realized at that time, however, that this migration of Bb may produce false labeling of neurons (see later). Bb-fluorescent glial nuclei also occur along axons which proceed from the area of the retrogradely labeled neurons to the injection area. They also occur along axons which proceed from the injection area to their terminations in other cell groups. It was therefore concluded that Bb proceeds both retrogradely and anterogradely through axons. However, after anterograde Bb transport through axons, no transynaptic labeling of recipient neurons was observed. Yet, it was later found that Bb [and nuclear yellow (NY), which is related to Bb], after very long survival times, may produce transynaptic neuronal labeling (Bentivoglio et al., 1980b; Aschoff and Holländer, 1982).

Propidium iodide produces retrograde labeling entirely different from that of Bb. Strongly PI-labeled neurons in SNC, after injection of PI (10% in water) in caudate putamen, display a brilliant orange-red fluorescence of cell body and proximal dendrites when viewed with filter-mirror system N2, but show little fluorescence when viewed with filter-mirror system A or D. PI-labeled neurons show very little nuclear labeling except for an orange-red fluorescence of the nucleolus. After long survival times relative to the transport distance (i.e., longer than the survival times in Table I), PI-fluorescent glial nuclei appear around the retrogradely labeled neurons.

Table I. Tracer Characteristics

		Tracer label			Survivai time necessary for proper labeling
Tracer	Tracer code number	Filter-mirror system A ^a	Filter-mirror system N2 ^b	Tracer solution(% w/v)	Rat ^c	Rat ^d	Rat ^e	Cat ^d	Cat ^e
Evans blue (EB)			Cytoplasm +nucleus: red	10	24–48 hr	I ^f	I	I	I
DAPI/primulin	Serva, Heidelberg18860/Eastman 1039	Cytoplasm + nucleus: light golden granules in cytoplasm, blue nucleus		2.5/10	2–4 d	I	I	I	I
Propidium iodide (PI)	Sigma P-5264		Cytoplasm + nucleolus: orange-red	3	2 d	7 d	7 d(light labeling)
Granular blue (GB)	Diamidino compound 186/134 ^g	Cytoplasm: blue with silver golden granules		5	2–4 d	5–7 d	7–9 d
True blue (TB)	Diamidino compound 150/129 ^g	Cytoplasm + nucleolus: blue		2	2–4 d	5–7 d	7–9 d	I	I
Fast blue (FB)	Diamidino compound 253/50 ^g	Cytoplasm: blue with fine silver granules		3	2d	4d	4d	3–4 w	3–4 w
Nuclear yellow (NY)	Benzimidazole compound Hoechst S76912 ^h	Nucleus + nucleolar ring: golden yellow		1	6 hr	24 hr	40 hr	±46 hr	±70 hr
Diamidino yellow (DY)	Diamidino compound 288/26 ^g	Nucleus + nucleolar ring: golden yellow (diffuse)		2	2–3 d	7d	10 d	3 w	4 w

a: Excitation light = 360 nm.
b: Excitation light = 550 nm.
c: Caudate putamen to nigra (SNC).
d: Spinal C5 to red nucleus.
e: Spinal T1 to cortex.
f: Inconsistent.
g: The code numbers were given by the Institute of Pharmacy and Food Chemistry of the Friedrich-Alexander University in Erlangen, Federal Republic of Germany, where these substances have been synthesized. For research purposes samples can be obtained from Prof. Dr. G. Illing, Warthweg 14–18, Postfach 1150, D-6114 Gross Umstadt, Federal Republic of Germany.
h: For research purposes, small samples of NY can be obtained from Dr. H. Loewe, Hoechst Aktiengesellschaft, Postfach 800320, 6230 Frankfurt am Main 80, Federal Republic of Germany.

From the characteristics of the retrograde labeling produced by Bb and PI, it was inferred that they could be used in double-labeling experiments demonstrating the existence of divergent axon collaterals. This was tested in the mammillary bodies of rats (Kuypers et al., 1979) by injecting Bb in one thalamus and PI in the other. Many PI–Bb double-labeled neurons were present in the lateral mammillary nucleus on both sides in the same manner as observed with EB and DAPI/Pr. These double-labeled neurons displayed an orange-red PI-fluorescent cytoplasm when viewed with filter-mirror system N2, and a yellow-green Bb-fluorescent nucleus when viewed with filter-mirror systems A and D. Since then, several investigators have used PI for double retrograde labeling and even for triple retrograde labeling of neurons. In addition, PI has been used in combination with histochemical and immunohistochemical techniques (De Olmos and Heimer, 1980; Van der Kooy and Wise, 1980; Björklund and Skagerberg, 1979a,b; Steinbusch et al., 1981; Brann and Emson, 1980; Hökfelt et al., 1979a,b, 1980). However, in our laboratory PI has seldom been used because it seems rather toxic, and lightly PI-labeled neurons are very difficult to distinguish from unlabeled ones. This latter difficulty, which is also encountered with EB, is due to the fact that even in a normal brain, many neurons, when viewed with filter-mirror system N2, display some orange-red autofluorescent granules in the cytoplasm. This makes lightly PI-labeled neurons difficult to distinguish from unlabeled ones, although PI-labeled neurons show an orange-red fluorescent nucleolus.

In our experiments, PI was not transported effectively over long distances, especially in cat (Kuypers et al., 1979). Therefore, the search for other retrograde tracers was continued. In view of the findings obtained with DAPI, special attention was paid to other diamidino compounds, all of which bind with RNA and DNA. A large series of diamidino compounds synthesized in Dr. O. Dann's laboratory were tested. Many of them gave retrograde fluorescent labeling of neurons and two compounds seemed useful as retrograde tracers, i.e., true blue (TB) ¹ and granular blue (GB) (Bentivoglio et al., 1979; Rosina et al., 1980). In rats, these compounds are transported effectively over long distances (from the spinal cord to the cerebral cortex) and produce a blue fluorescent labeling of the neuronal cytoplasm in cell body and proximal dendrites when viewed with filter-mirror system A. True blue also gives a pronounced blue labeling of the nucleolus, and granular blue produces an accumulation of silver-blue granules in the cytoplasm (Figs. 2 and 3). Moreover, after long survival times relative to the transport distance (8 days, cf. Table I) in the nigrostriatal system, some blue fluorescent glial nuclei are present around the retrogradely labeled neurons. These and further findings have shown that TB is a very effective retrograde tracer in double- and triple-labeling experiments in rat (De Olmos and Heimer, 1980; Swanson et al., 1980; Swanson and Kuypers, 1980a,b).

True blue, despite its very favorable characteristics as a retrograde tracer, has the disadvantage that it is not transported effectively over long distances in cat. However, another "blue" diamidino compound was found, i.e., "fast blue" (FB) (Bentivoglio et al., 1980a), which is more soluble in water and is rather effectively transported retrogradely over long distances in rat, cat, and monkey (Kuypers et al., 1980; Huisman et al., 1982). Retrogradely FB-labeled neurons display a blue fluorescent cytoplasm when viewed with filter-mirror system A, but the FB fluorescence is a little duller and slightly more grayish than the TB fluorescence (Fig. 4). After long survival times (much longer than cited in Table I), the blue cytoplasm contains some orange fluorescent granules, and glial nuclei surrounding the labeled neurons become fluorescent. Moreover, contrary to the findings with TB, some very heavily FB-labeled neurons with a brightly blue fluorescent cytoplasm occasionally display a white-to-bluish fluorescent nucleus (Bharos et al., 1981; A. M. Huisman et al., unpublished observations). In the central nervous system this phenomenon was rarely observed, and only when a relatively long survival time was combined with a short transport distance. However, in the peripheral nervous system this phenomenon is apparently more frequently observed (M. Illert, personal communication) (see later).

The labeling characteristics of the "blue" tracers suggested that Bb could be combined with TB or FB in double-labeling experiments because the double-labeled neurons, when viewed in filter-mirror system A, would show a blue fluorescent cytoplasm and a yellow fluorescent nucleus. In addition, in these cases, the neuronal autofluorescence would not interfere with the detection of the tracer fluorescence because, with filter-mirror system A, the autofluorescence appears as brown-yellow granules in the blue-labeled cytoplasm. The effectiveness of such double labeling was demonstrated in experiments using the efferent connections of the mammillary bodies in rat and cat as a model (Kuypers et al., 1980). However, Bb also provides a yellow-green cytoplasmic labeling which might obscure the blue TB or FB labeling. Therefore, in such double-labeling experiments, another benzimidazole (NY, provided by Dr. H. Loewe of the Hoechst Company) is regularly used (Bentivoglio et al., 1980a), and gives primarily a nuclear labeling similar to that obtained with Bb (Figs. 2, 3, and 4). With respect to this type of double labeling it may be argued that the combination of FB and NY has the disadvantage that, in some cases, single-FB-labeled neurons show some blue-to-white FB labeling of the nucleus, which could make such neurons difficult to distinguish from FB–NY double-labeled neurons. However, in our experiments using brain, only very seldom was a strong FB labeling of the neuronal nucleus obtained (Bharos et al., 1981; Huisman et al., unpublished observations), and this blue-to-white FB labeling could always be clearly distinguished from the yellow-to-green NY (or DY, see later) labeling. Therefore, the combination of FB and NY has been consistently used in double-labeling experiments in cat and monkey (Figs. 1 and 4), and the combination FB, NY, and EB has been used in triple-labeling experiments in rat (Bentivoglio and Molinari, 1982).

In one of the first studies in which the TB–NY and TB–Bb combination was used, it became obvious, however, that the NY and Bb migration out of the retrogradely labeled neurons might give rise to false labeling of neurons. The aim of this particular study was to determine whether corticospinal neurons in rat possess callosal collaterals (Catsman-Berrevoets et al., 1980). For this purpose NY (10% in water) was injected in one hemisphere and TB (2% in water) was injected ipsilaterally in the spinal cord, followed by approximately 6 days survival time. In these experiments, many of the TB-Iabeled corticospinal neurons in the noninjected hemisphere were TB–NY double labeled. However, in electrophysiological experiments (Catsman-Berrevoets et al., 1980), no indication of the existence of corticospinal callosal collaterals could be obtained. Moreover, with other tracer combinations, e.g., EB and GB, no unequivocal double labeling of corticospinal neurons was found. It was therefore concluded that in the TB–NY experiments, some false NY labeling of corticospinal neurons had occurred, probably due to migration of NY out of the retrogradely labeled callosal neurons, as indicated by the presence of NY-labeled fluorescent glial nuclei around these neurons (cf. Kuzuhara et al., 1980). In a new set of experiments (Bentivoglio et al., 1980b) it was found that the NY and Bb migration out of retrogradely labeled neurons occurs gradually during the survival period, such that the nucleus is labeled first, and then the cytoplasm plus the surrounding glial nuclei, which after longer survival times become progressively more brilliantly fluorescent. These experiments further showed that the migration of Bb and NY from the retrogradely labeled neurons may be prevented by using 1% Bb or NY in water (instead of 10%) and by restricting the survival times, such that the glial nuclei around the retrogradely labeled neurons are either nonfluorescent or show only dull fluorescence, i.e., much duller than the retrogradely labeled neurons. When, in rat, the injections in the spinal cord and the hemisphere were repeated in this manner such that TB was first injected in the spinal cord followed by a 6-day survival time, and NY (1% in water) was later injected in the hemisphere at 28 hr before the animal was killed (28-hr NY survival time), none of the corticospinal neurons was double labeled. However, this was not due to a failure of the TB–NY combination to double label the neurons because, when this same procedure was applied to the mammillothalamic connections, many double-labeled neurons were present in the lateral mammillary nucleus even after very short NY survival times. In restricting the survival times, NY was preferred over Bb because NY requires a slightly longer transport time than Bb and migrates more slowly than Bb out of the retrogradely labeled neurons, thus allowing a somewhat longer survival time.

The necessity to inject the two tracers at different times during the survival period, however, makes the double labeling with TB or FB in combination with NY a somewhat cumbersome procedure. Nevertheless, the combination of NY with TB or FB (Figs. 1, 3, and 4) is still preferred over the combination of red and blue tracers because, as pointed out previously, when using the red tracers, lightly labeled neurons are difficult to distinguish from nonlabeled neurons.

The complication that TB or FB and NY have to be injected at different times during the survival period could be avoided if NY could be replaced by another tracer which would also label mainly the nucleus, but would not migrate out of the retrogradely labeled neurons. Such a tracer could be injected together with TB or FB in the same session. In order to find such a tracer, several diamidino compounds synthesized in Dr. O. Dann's laboratory were tested. The diamidino compound No. 28826 was found and named diamidino yellow dihydrochloride (DY·2HCl). ² This compound, which is related to GB, TB, and FB, appears to meet the requirements for labeling and migration and will soon be commercially available (Keizer et al., 1983). At a 360-nm excitation wavelength, DY·2HCl gives a diffuse, golden-yellow labeling of the neuronal nucleus with a lightly fluorescent ring around the nucleolus, and a diffuse, yellow labeling of the cytoplasm, which sometimes also contains golden-yellow fluorescent granules (Figs. 2 and 5). DY·2HCl is effectively transported over long distances in rat and cat (Fig. 1), and can be successfully combined with TB and FB in double-labeling experiments aimed at demonstrating the existence of divergent axon collaterals (Keizer et al., 1983).

When trying to obtain double labeling of neurons by way of divergent axon collaterals, it should be realized that the number of double-labeled neurons is determined by the sensitivity of the weakest tracer. Therefore, a combination of the most effective tracers should be used, because if one tracer is much less sensitive than the other, i.e., labels far fewer neurons, only a small percentage of the neurons giving rise to divergent axon collaterals will be double labeled. In light of this, the combination of TB or FB and NY or DY is preferred.

The fluorescence and the histological features produced in the injection areas by the various tracers reported in this article (EB, DAPI/Pr, PI, GB, TB, FB, Bb, NY, and DY) have been described (Kuypers et al., 1977, 1979, 1980; Bentivoglio et al., 1979a,b, 1980a,b; Catsman-Berrevoets and Kuypers, 1981; Keizer et al., 1983). These injection areas in general consist of several concentric fluorescent zones which surround the end of the needle track where the tracer has been deposited in the brain tissue. The findings in various experiments indicate that the uptake and the retrograde axonal transport of the different tracers occur primarily from the central zones of the injection areas, from fiber termination areas as well as from broken axons. However, as previously mentioned, SITS seems to be taken up only by terminals, and not by broken axons (Schmued and Swanson, 1982).

The injection of the tracers produces tissue necrosis in the center of the injection area. This is most pronounced with DAPI, GB, TB, FB, and DY·2HCl. However, the occurrence of such necrosis does not interfere with the retrograde axonal transport of the tracers, as indicated by the fact that DY·2HCl, as TB, produces considerable necrosis in the center of the injection area but frequently gives a retrograde labeling of a larger number of neurons than NY, which produces relatively little necrosis.

The preceding description of the retrograde neuronal labeling obtained with different tracers is based on observations in frozen-section material of the brain. In this respect, it is of importance to emphasize that in different parts of the central nervous system, and after different histological procedures, a different type of labeling may be obtained. For example, as described by Björklund and Skagerberg (1979a,b) using freeze-dried material, TB and PI are present in the neuronal cytoplasm as ice blue and red fluorescent granules instead of being distributed diffusely, as observed in frozen-section material (Fig. 6). Further, under certain conditions, the type of retrograde labeling of motoneurons observed in frozen-section material after transport of the tracers through peripheral nerves (Illert et al., 1982) differs from that obtained in neuronal systems in the brain. For example, after dipping branches of cat radial nerve in 10% FB dissolved in ethylene glycol (Illert et al., 1982), a bright blue fluorescent labeling of both the motoneuronal cytoplasm and the motoneuronal nucleus was obtained which was so bright that it could be observed under low magnification (10× objective). Moreover, after intramuscular injections of 2% NY dissolved in ethylene glycol, a strong, predominantly white-blue cytoplasmic NY labeling of motoneurons occurred which could also be observed under low magnification, while only a soft, yellow NY labeling of the nucleus was present (Illert et al., 1982). Thus, under those specific circumstances, NY-labeled motoneurons resemble FB-labeled motoneurons. This has led Illert and co-workers to the conclusion that, when trying to differentially label motoneurons from different peripheral nerves using their procedure, tracers with different emission spectra should be used (Fig. 7). The unusual FB, NY, and Bb retrograde labeling obtained in motoneurons by Illert and co-workers is probably due to the fact that, in following their procedure, the neurons are almost entirely saturated by the tracers. This probably results from the fact that large quantities of the tracers are injected in the muscles and that the peripheral nerves are dipped in high concentrations of tracers dissolved in ethylene glycol. This is indicated by the observations of R. N. Lemon and R. B. Muir (personal communications), who obtained an FB motoneuronal labeling similar to that observed in neuronal systems in the brain after dipping the deep branch of monkey ulnar nerve in 5% FB (instead of 10%) dissolved in water with 2% dimethyl sulfoxide (DMSO) (instead of ethylene glycol).

The large intramuscular injections as used by Illert and co-workers produced not only very heavy labeling of the motoneurons, but also labeling of the endothelial cells lining the blood vessels in the spinal cord. This endothelial labeling is only very rarely observed after small injections in the central nervous system and, in all likelihood, is due to the fact that the dye had entered the bloodstream.

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Antimicrobial and cytotoxic activity of graphene-based perioceuticals

Marcela Popa , ... Veronica Lazăr , in Fullerens, Graphenes and Nanotubes, 2018

14.5 The Cytotoxicity Assay

HT29 cells were stained with PI 100 μg/mL. Data acquisition and analysis were performed to obtain histograms representing the DNA content using the Cell Cycle program on a Coulter EPICS XL Beckman Coulter Flow Cytometer. To measure the DNA content of the PI-stained nuclei, histogram type graphs were used. Excitation was 488 nm. Both the peak and the integrated fluorescence signal area were purchased. 50,000 events have been purchased and analyzed.

Cell cycle analysis involves determining the number of cells in a population in each of its phases (G0, G1, S, G2, M) by measuring the amount of DNA contained in the nucleus (Fig. 14.3).

Analysis of the cytotoxic effect of proposed transport and release systems, based on graphite functionalized with Ag and loaded with essential clove oil, showed that they have high biocompatibility and are not cytotoxic. Of the tested variants, variant 2, represented by graphite and cloves, at a concentration of 10 μL/mL induced a decrease in the S phase cells number, which could suggest the antiproliferative effect of this combination (Fig. 14.4).

In the context of increased rates of antibiotic resistance, the discovery of active agents against bacteria embedded in biofilm represents an important control strategy for chronic oral diseases (Projan and Youngman, 2002). The antimicrobial susceptibility testing results of newly developed materials, consisting of graphenes functionalized with AgNPs and clove oil, are indicating that they can be used as alternative strategies for controlling periopathogens.

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Apoptosis

Zbigniew Darzynkiewicz , Elzbieta Bedner , in Methods in Enzymology, 2000

Results

Apoptotic cells have a decrased PI (or DAPI) fluorescence compared with the cells in the main peak (G₁) (Fig. 6). It should be emphasized that the degree of extraction of low molecular weight DNA from apoptotic cells, and consequently the content of DNA remaining in the cell for flow cytometric analysis, may vary dramatically depending on the degree of DNA degradation (duration of apoptosis), the number of cell washings, and the molarity of the washing and staining buffers. Therefore, in step 3, less or no extraction buffer need be added (e.g., 0–0.2 ml) if DNA degradation in apoptotic cells is extensive (late apoptosis) and more should be added (up to 1.0 ml) if DNA is not markedly degraded (early apoptosis), and there are problems with separating apoptotic cells from G₁ cells because of their overlap on DNA content frequency histograms.

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hollingsworthporser.blogspot.com

Source: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/propidium-iodide

Blue Strip of Dna Came From What Cell

Propidium Iodide

Tumor Immunology and Immunotherapy – Molecular Methods

5 Cell-cycle arrest analysis

5.1 Equipment, reagents and software

5.2 Steps and annotations

5.2.1 Cell culture

5.2.2 PI staining solution

5.2.3 Fixation and staining with PI staining solution

5.2.4 Using flow cytometry instrument and data analysis

5.2.5 Using markers for different phases of cell-cycle

Primary Cultures of Cardiac Myocytes as In Vitro Models for Pharmacological and Toxicological Assessments

1 Propidium iodide dye exclusion assay

Cell Biological Applications of Confocal Microscopy

G Nuclear Stains

Acknowledgments

Monitoring vesicular trafficking in cellular responses to stress - Part A

2.2.1 Materials, reagents and equipment

Nanomedicine

4.3.1 Protocol III

4.3.1.1 Determination of apoptosis versus necrosis

Apoptosis

Reagents

Mesoporous Silica-based Nanomaterials and Biomedical Applications, Part B

4.1.1 Macroscopic and Multimodal Imaging of Gastrointestinal Tract

Advances in Cellular Neurobiology

II Development of the Multiple Retrograde Fluorescent Tracer Technique for Demonstrating Axon Collaterals

Antimicrobial and cytotoxic activity of graphene-based perioceuticals

14.5 The Cytotoxicity Assay

Apoptosis

Results

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