Background: Simultaneous use of cell-permeant and impermeant fluorescent nuclear dyes is a common method to study cell viability and cell death progression. Although these assays are usually conducted as end-point studies, time-lapse imaging offers a powerful technique to distinguish temporal changes in cell viability at single-cell resolution. SYTO 13 and Hoechst 33342 are two commonly used cell-permeant nuclear dyes; however their suitability for live imaging has not been well characterized. We compare end-point assays with time-lapse imaging studies over a 6 h period to evaluate the compatibility of these two dyes with longitudinal imaging, using both control neurons and an apoptotic neuron model.
Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen of macrophages that causes a chron- ic enteritis (Johne’s disease) in ruminants. The purpose of this study was to determine whether M. avium subsp. paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with M. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because mono- cytes infected with heat-killed M. avium subsp. paratuberculosis did not exhibit a significant increase in apo- ptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp. para- tuberculosis did not significantly alter the number of apoptotic cells.
Thus, we can conclude that polarity of the environ- ment around fluorescent dye Hoechst 33342 is almost unchanged while it sorption on the surface of HP1, t- RNA or DNA. At small concentrations, Hoechst binds mainly at the surface of DNA, RNA or HP1. At small concentrations, it has no any specificity to certain nu- cleotides. In the case of unwound sites of DNA or hair- pins, it binds inside, but without stacking with nucleo- tides. The energy transfer from nucleotides to Hoechst is absent due to remoteness of Hoechst from nucleotide chromophores and also of their “bad” non-stacking ori- entation. The mutual quenching of emission of Hoechst by actinomycin D (AMD) and, vice versa, of emission of 7-amino-actinomycin D (7AAMD) by Hoechst in DNA and HP1 is observed. It is due to two main reasons: dy- namic deactivation and mutual replacing in binding sites.
Unless otherwise specified, experiments were performed in quadruplicate parallel instances, and data were analyzed with the R software (http://www.r-project.org/). The first-line flow cytometry data analysis was performed using the flowcore package for R (http://www.bioconductor. org) upon gating on events with normal forward and side scatter parameters. Microscopy images were analyzed by means of the MetaXpress (Molecular Devices) software. In particular, images were segmented using the built-in custom module editor to identify nuclei (based on Hoechst 33342 fluorescence), and cytoplasmic regions (based on DiOC 6 (3)
Lichens are complex organisms living in a symbiotic relationship with fungi and algae have recently received special interest in cancer research. The cytotoxic activities of Usnea filipendula Stirt. lichen extract was investigated on colon cancer cell lines, HCT-15 and HT-29. Sulphorhodamine B and ATP cell viability tests were used to monitor cytotoxic activity. The mode of cell death (apoptosis/necrosis) was determined using caspase-cleaved cytokeratin 18 (M30), caspase-3/7 activity and fluorescence staining techniques that included, Annexin-V, Hoechst 33342 and propidium iodide. Usnea filipendula showed dose and time- dependent antiproliferative effect in HCT-15 and HT-29 cells. The IC 50 values in HCT-15 and HT-29 cells were 17.92 and 41.87 µg/ml, respectively. The extract induced apoptosis in both cell lines especially in HCT-15 cells in which caspase-3/7 activity was increased. Usnea filipendula was cytotoxic to colon cancer HCT-15 and HT-29 cell lines by inducing early or late apoptosis as evidenced by translocation of phosphatidylserine, pyknotic nuclei and nuclear condensation. Further studies would help to understand the full potential of Usnea filipendula as a novel anticancer therapy.
semen cooled to 5 °C for 6 h. After cooling the semen was packaged into 0.5 mL straws cooled to −120 °C for 10 min before being plunged into liquid nitrogen for storage using static nitrogen vapor. For this study, the frozen sperms were thawed in a water bath at 37 °C for 45 s. The first prepared sperm samples were fluorescent- stained, and evaluated for viability and acrosome damage using flow cytometry (FACS Aria II, BD Biosciences, San Jose, CA, USA) and the staining patterns were verified by inspecting sperm samples under an epifluorescence microscope (Olympus, BX5, Tokyo, Japan). We used a dual blue/green filter set. The second prepared sperm samples were dyed with Hoechst 33342 and separated by
From our data, we can conclude that for these specimens the photo-bleaching rate scales faster than linear with the excita- tion power, but not with an order (n) higher than 2. This is in contrast to the literature finding orders of n > 3 for several flu- orophores (Patterson & Piston, 2000; Kalies et al., 2011) and n = 2 for Hoechst 33342 (Kalies et al., 2011). The fact that the photo-bleaching rate is wavelength-dependent and increases at shorter excitation wavelengths is, however, in agreement with Patterson & Piston (2000) and Bush et al. (2007). Also, in agreement with Patterson & Piston (2000) and Bush et al. (2007), we observe that the photo-bleaching rate is not simply increased for a less efficient excitation wavelength, where the number of excess photons that can lead to further absorption from the excited state is large, nor does it scale with the num- ber of excitation events, that is, the photo-bleaching rates in Figure 3(A) are not proportional to the excitation efficiency
H. odorata extract (250 µ g/mL) was used to treat HepG2 cells for 48 hours, and then, treated cells were stained with Hoechst 33342 to assess nuclei of all cells and with PI to assess nuclei of dead cells only. Control cells were treated with 0.5% methanol solvent, which is the same concentration of methanol solvent in H. odorata extract-treated samples. H. odorata-treated cells appear like dead cells with disin- tegration of nuclei (Figure 6A, C, and E). The cells also exhibited nuclei condensation and appeared to have brighter staining than the surrounding live cells. Conversely, control cells stained with Hoechst 33342 but not with PI; this means that there was no death cell in the control cells (HFs) when compared to treated HepG2 cells (Figure 6B, D, and F).
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Tissues were embedded in paraffin then cut in thin sec- tions and preserved for further staining. The sections were then deparaffinized in xylene followed by rehydra- tion through graded ethanol/water until rehydrated in phosphate buffered saline (PBS). Sections were then blocked in 5% normal goat serum containing 0.1% Tri- ton X-100 in PBS. They were then incubated in blocking solution containing anti-YAP and anti-Scribble antibody overnight at 4°C. They were then washed 3 times in PBS followed by incubation with fluorescently labeled sec- ondary antibodies for 1 h at room temperature. Sections were extensively washed in PBS, incubated in Hoechst 33342 (Life Science Technologies), suspended in PBS prior to mounting onto slides and imaging. All images were captured on a Leica DM6100 inverted microscope with appropriate filter sets using Intelligent Imaging In- novations (3i) software for acquisition. All post hoc im- aging was done with Photoshop 6.0.
The cellular uptake of micelles was observed using a Zeiss 510 Meta Confocal microscope. A 488 nm wavelength laser was used to excite the Oregon-green (excitation/emission maxima = 495/521 nm) and CellMask™ deep red plasma membrane stain (excitation/emission maxima = 649/666) was excited with a laser of 633 nm wavelength. The Hoechst 33342 stain (excitation/emission maxima = 350/461) was illuminated with a 100-watt high-pressure mercury plasma arc-discharge lamp (HBO 100). The control untreated cells (without micelles) were used to subtract the background or auto fluorescence.
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Since agents that induce latent HIV-1 through global T cell activation are toxic, we confirmed that disulfiram does not cause global T cell activation. Increased cell size is a direct indicator of T cell activation. Unlike cells stimulated with anti- CD3 plus anti-CD28 antibodies, latently infected cells treated with disulfiram retained the same small size as untreated cells (Fig. 3A). By staining for well-characterized activation mark- ers, we confirmed that neither disulfiram nor DDTC induced T cell activation. Freshly isolated primary resting CD4 ⫹ T cells were treated with different concentrations of disulfiram, DDTC, or anti-CD3 plus anti-CD28 for 24 h and then stained for surface expression of CD69, CD25, and HLA-DR. Neither disulfiram nor DDTC upregulated surface expression of these markers, except for a minor induction of CD69 (Fig. 3B). In addition, no change was observed in surface expression of CCR7 (data not shown). Neither disulfiram nor DDTC in- duced changes in global DNA or RNA levels as measured by Hoechst 33342/pyronin Y staining, indicating that disulfiram treatment does not stimulate proliferation in primary resting CD4 ⫹ T cells (Fig. 3C).
A. Dose-response curve for Jurkat cells treated with various concentrations of either AMD4 or AMD5 for 48 hrs. Cells were stained with cell-permeable Hoechst 33342 dye; brightly stained and condensed nuclei were considered to be apoptotic while non-bright, smooth nuclei were considered healthy (non-apoptotic). The degree of apoptosis was calculated by the number of apoptotic cells counted over the total number of cells visible and displayed as a percentage. A minimum of 5 fields with at least 100 cells per field were counted and tabulated using Microsoft Excel software; values that are statistically sig- nificant to p < 0.05 are indicated with an asterisk. Micromolar is represented as uM in this figure only. B. Time Course: A measurement of the degree of apoptosis induced in Jurkat cells treated with 10 μ M of either AMD4 or AMD5 over 72 hours. Following treatment, Jurkat cells were incubated with cell-permeable Hoechst 33342 dye; brightly stained condensed nuclei were considered to be apoptotic while non-bright, smooth nuclei were considered healthy. The degree of apoptosis was calculated by the number of apoptotic cells counted over the total number of cells visible displayed as a percent- age. A minimum of 5 fields with at least 100 cells per field were counted and tabulated using Microsoft Excel software; values that are statistically significant to p < 0.05 are indicated with an asterisk.
Stem cells can be purified based on the efflux of fluor- escent dyes such as rhodamine 123 (rho123) and Hoechst 33342 (Hoechst). A widely used flow cytometry assay for identifying stem cells defines a side population (SP) of cells displaying low Hoechst fluorescence and comprising about 0.05% of total cells . This SP popu- lation is highly enriched for lineage-specific stem cells. The dye efflux component of the SP phenotype has been assumed to express ATP-binding cassette (ABC) trans- porters such as P-glycoprotein (P-gp), encoded by the multidrug resistance 1 (MDR1) gene, and breast cancer resistance protein (BCRP)/ABCG2. BCRP expels Hoechst but not rho123, while P-gp expels both Hoechst and rho123. Accordingly, normal and cancer stem cells express high levels of P-gp and BCRP. These two ABC transporters and multidrug resistance-associated protein 1 (MRP1)/ABCC1 constitute the three principle ABC transporters implicated in multidrug resistance. MRP1 has also been found to be expressed in SP cells .
Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib. It was shown in vitro by Western blot, flow cytometry, and ELISA that bortezomib accumulated HIF-1` in non-functional forms and blocks its hypoxia response in human colorectal cancer cell lines. Ex vivo experiments were performed with fluorescent immunohistochemical staining techniques using multiple endogenous and exogenous markers to identify hypoxia (pimonidazole, HRE-TKeGFP), blood flow/ permeability (Hoechst 33342), micro-vessels (CD31 and SMA), apoptosis (cleaved caspase 3) and hypoxia response (CA9). After bortezomib administration, overall apoptosis index was significantly increased and blood perfusion was dramatically decreased in tumor xenografts. More importantly, apoptosis signals were found preferentially located in moderate and severe pretreatment hypoxic regions in both tumor and endothelial cells. Meanwhile, DCE MRI examinations showed that the tumor blood flow and permeability decreased significantly after bortezomib administration. The present study revealed that bortezomib reduces tumor hypoxia response and blood perfusion, thus, presenting antivascular properties. It will be important to determine the hypoxic/perfusion status pre- and during treatment at further translational studies.
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T cells preferentially migrate into the tumor stroma. (A) Preactivated T cells (Hoechst; green) were added to a human lung tumor slice that was subsequently stained for fibronectin and EpCAM to respectively identify stromal (red) and tumor epithelial cell (blue) regions. Each image, captured with a widefield microscope, is the maximum projection of 4 images spanning 60 μm in the z direction beneath the cut surface of the slice. (B) Concentration of in vitro activated T cells and resident CD3 + cells in the stromal and tumor cell regions. (C) Concentration of in vitro
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Based on the method of 15 the cell pathology was detected by staining of trypsinized cells (5X 10 5 /ml) with 1 µl of Hoechst 33258 (1 mg/ml, aqueous) for 10 min at 37ºC. A drop of cell suspension was placed on a glass slide and a cover slip was laid over to reduce light diffraction and cell pathology were observed using a fluorescent microscope (Axio Scope A1, Carl Zeiss, Germany) fitted with a 377-355 nm filter and the cells reflecting pathological changes were observed and photographed.
The present study aims to investigate the anti-proliferative, apoptotic properties of prodigiosin, using a human oral squamous cell carcinoma HSC-2 cell line as a model system. HSC- 2 cells were cultured in the presence of prodigiosin at various concentrations (1–30µg/ml) for 48 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that prodigiosin inhibited the cells viability in time and concentration dependent characteristics at different concentrations. We found that anti-proliferative effect of prodigiosin was associated with apoptosis on HSC-2 cells by determinations of DNA fragmentation, Hoechst 33258 staining, caspases activity, and TNF-α was significantly changed when compare DMSO control group. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with prodigiosin at various concentrations and times. These results suggested that prodigiosin treatment inhibited proliferation and induction of apoptosis in human oral squamous cell carcinoma HSC-2.
Day 4: Immunoﬂuorescence staining and imaging. Staining procedures were continued in the following order with each antibody left on for 1.5 h: AlexaFluor 647 goat anti-mouse (ﬁnal concentration 2 μg ml 1 ) to detect YAP/TAZ, rat primary anti α-tubulin antibody (ﬁnal concentration 1 μg ml 1 ), AlexaFluor 568 goat anti-rat to detect α-tubulin (ﬁnal concentration 2 μg ml 1 ) and Phalloidin 488 combined in the same step. Finally the nuclear stain Hoechst (Sigma Aldrich, cat # 33,258, ﬁnal concentration 10 μg ml 1 ) was added for 15 min, upon which it was removed and 15 μl of PBS were dispensed. All cells were imaged using an automated Opera Quadruple Enhanced High Sensitivity (QEHS) spinning-disk confocal microscope (PerkinElmer) with 20x air lens. Lasers and their corresponding ﬁlters used were: 405(450/50), 561 (600/40), 488 (540/751), 640 (690/50).
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Cells were treated with various concentrations of TW9183 , the medium was removed 48 h after incubation, cells were fixed with 100 µl of 4% paraformaldehyde each well, washed twice with PBS, and stained with 80 µl Hoechst 33342 for 10 min at room temperature, removed the staining solution, washed with PBS twice, observed under a fluorescence microscopy [13-14].