Propidium Iodide Fluorescence Intensity Research Articles

Antibacterial activity and mechanism of cinnamon essential oil nanoemulsion against Pseudomonas deceptionensis CM2

This work aimed to evaluate the antibacterial activity and mechanism of cinnamon essential oil nanoemulsion (CON) against Pseudomonas deceptionensis CM2. The results revealed that CON could effectively inhibit the proliferation of P. deceptionensis CM2 cells in a time- and concentration-dependent manner. After 4 h of incubation with CON at the minimum inhibitory concentration (0.125 mg/mL), the relative fluorescence intensity of propidium iodide and 1-N-phenylnapthylamine (NPN) was increased by 32.0% and 351.4%, respectively. The membrane permeability of P. deceptionensis CM2 cells was significantly disrupted after CON treatment, resulting in the leakage of intracellular substances (such as proteins and electrolytes). CON also caused significant increases in the DiBAC4(3) fluorescence intensity of P. deceptionensis CM2 cells. These results demonstrate that CON induced inactivation of P. deceptionensis CM2 by destroying the integrity and function of bacterial membrane. A higher level of intracellular reactive oxygen species (ROS) was observed in CON-treated cells (p < 0.05), compared with control cells. Moreover, the addition of glutathione to the growth medium remarkably decreased the antimicrobial activity of CON against P. deceptionensis CM2, further confirming that oxidative stress played an important role in the antimicrobial activity of CON. Overall, CON may exhibit antibacterial effects by causing damage to the bacterial membranes and oxidative stress.

Combined Application of Aminoglycosides and Ascorbic Acid in the Elimination of Proteus mirabilis Rods Responsible for Causing Catheter-Associated Urinary Tract Infections (CAUTIs)-A Molecular Approach.

Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTIs). In this study, we verified the effectiveness of amikacin or gentamicin and ascorbic acid (AA) co-therapy in eliminating uropathogenic cells, as well as searched for the molecular basis of AA activity by applying chromatographic and fluorescent techniques. Under simulated physiological conditions, a combined activity of the antibiotic and AA supported the growth (threefold) of the P. mirabilis C12 strain, but reduced catheter colonization (≤30%) in comparison to the drug monotherapy. Slight modifications in the phospholipid and fatty acid profiles, as well as limited (≤62%) 2',7'-dichlorofluorescein fluorescence, corresponding to the hydroxyl radical level, allowed for the exclusion of the hypothesis that the anti-biofilm effect of AA was related to membrane perturbations of the C12 strain. However, the reduced (≤20%) fluorescence intensity of propidium iodide, as a result of a decrease in membrane permeability, may be evidence of P. mirabilis cell defense against AA activity. Quantitative analyses of ascorbic acid over time with a simultaneous measurement of the pH values proved that AA can be an effective urine acidifier, provided that it is devoid of the presence of urease-positive cells. Therefore, it could be useful in a prevention of recurrent CAUTIs, rather than in their treatment.

Evaluating Alternate Methods of Determining the Antimicrobial Efficacy of Contact Lens Care Products against Acanthamoeba Trophozoites

Acanthamoeba keratitis (AK) is a serious ocular infection caused by a ubiquitous free-living amoeba, Acanthamoeba. This infection often results in extensive corneal damage and blindness, and is notoriously difficult to cure. While Acanthamoeba is an abundant organism, AK is most associated with contact lens hygiene noncompliance and inadequate contact lens care (CLC) disinfection regimens. Thus, accurate and timely antimicrobial efficacy testing of CLC solutions is paramount. Published methods for antimicrobial efficacy testing of Acanthamoeba trophozoites requires 14 days for results. Presently, alternate and/or rapid methods for evaluating CLC products rarely demonstrate equivalent results compared to commonly-reported methods. Propidium iodide is a cellular stain that can only bind to cells with damaged outer membranes. We evaluated propidium iodide staining as an alternative method for determining the relative antimicrobial efficacy of 11 different CLC products against Acanthamoeba trophozoites. Following exposure to a CLC product, the fluorescence intensity of propidium iodide in an Acanthamoeba population demonstrated a strong correlation to the log reduction determined by established, growth-based Acanthamoeba testing used to evaluate the antimicrobial efficacy of CLC products. Thus, propidium iodide was found to be an effective rapid tool for determining cell death in Acanthamoeba trophozoites following exposure to CLC solutions.

Rapid and cost-effective evaluation of bacterial viability using fluorescence spectroscopy

A rapid and easy method that takes advantage of an inexpensive and portable fibre-based spectroscopic system (optrode) to determine the ratio of live to dead bacteria is proposed. Mixtures of live and dead Escherichia coli with proportions of live:dead cells varying from 0 to 100% were stained using SYTO 9 and propidium iodide (PI) and measured using the optrode. We demonstrated several approaches to obtaining the proportions of live:dead E. coli in a mixture of both live and dead, from analyses of the fluorescence spectra collected by the optrode. To find a suitable technique for predicting the percentage of live bacteria in a sample, four analysis methods were assessed and compared: SYTO 9:PI fluorescence intensity ratio, an adjusted fluorescence intensity ratio, single-spectrum support vector regression (SVR) and multi-spectra SVR. Of the four analysis methods, multi-spectra SVR obtained the most reliable results and was able to predict the percentage of live bacteria in 108 bacteria/mL samples between c. 7 and 100% live, and in 107 bacteria/mL samples between c. 7 and 73% live. By demonstrating the use of multi-spectra SVR and the optrode to monitor E. coli viability, we raise points of consideration for spectroscopic analysis of SYTO 9 and PI and aim to lay the foundation for future work that uses similar methods for different bacterial species.

Wound Healing Potential of the Standardized Extract of Boswellia serrata on Experimental Diabetic Foot Ulcer via Inhibition of Inflammatory, Angiogenetic and Apoptotic Markers

The aim of the present study was to evaluate the wound healing potential and possible mechanism of action of the standardized extract of Boswellia serrata against the experimental model of diabetic foot ulcer. α-Boswellic acid was isolated from the standardized extract of B. serrata and characterized (HPLC, 1H-NMR, 13C-NMR, ESI-MS). Diabetes was induced in Sprague-Dawley rats by streptozotocin (55 mg/kg, i. p.), and wounds were created on the dorsal surface of the hind paw. B. serrata (100, 200, and 400 mg/kg, p. o.) was administered to the rats for 16 days. The HPLC analysis showed a single peak with a retention time of 12.51 min. The compound was identified with ESI-MS [M + Na]+ = 455.37 as α-boswellic acid. Treatment with B. serrata (200 and 400 mg/kg) significantly increased the rate of wound contraction via modulation of oxido-nitrosative stress and elevated the hydroxyproline level at the wound area. reverse transcription-PCR analysis revealed that streptozotocin-induced increases in TNF-α, interleukin-1β, interleukin-6, nuclear factor-kappa-light-chain-enhancer of activated B cells, and Bcl-2-associated X protein, and decreases in angiopoietin-1, Tie2, transforming growth factor beta 1, vascular endothelial growth factor, and collagen-1 mRNA expression were significantly inhibited by B. serrata. It also significantly reduced wound cellular necrosis as evaluated by flow cytometry using propidium iodide fluorescence intensity. Streptozotocin-induced histopathological alterations were also significantly ameliorated by B. serrata. In conclusion, standardized extracts of B. serrata exert its wound healing potential via orchestrating mechanisms, which include the inhibition of oxido-inflammatory markers (oxido-nitrosative stress, TNF-α, interleukins, and nuclear factor-kappa-light-chain-enhancer of activated B cells), increased collagen synthesis (hydroxyproline and collagen-1) and angiogenesis (Ang-1/Tie2), promoting growth factors (transforming growth factor beta 1 and vascular endothelial growth factor), and inhibition of apoptosis (Bcl-2-associated X protein) to accelerate wound healing in experimental delayed diabetic foot ulcer.

Polyethylene glycol molecular weight influences the ClearT2 optical clearing method for spheroids imaging by confocal laser scanning microscopy.

Some fluorescence microscopy techniques, such as confocal laser scanning microscopy (CLSM), have a limited penetration depth. Consequently, the visualization and imaging of three-dimensional (3-D) cell cultures, such as spheroids, using these methods can be a significant challenge. Therefore, to improve the imaging of 3-D tissues, optical clearing methods have been optimized to render transparency to the opaque spheroids. The influence of the polyethylene glycol (PEG) molecular weight (MW) used in the ClearT2 method for the imaging of propidium iodide (PI)-stained spheroids was investigated. The results demonstrated that the ClearT2 clearing method contributes to spheroids transparency and to the preservation of PI fluorescence intensity for all the PEG MW used (4000, 8000, and 10,000Da). Furthermore, the ClearT2 method performed using PEG 4000Da allowed a better PI signal penetration depth and cross-section depth. Overall, the optimization of PEG MW can improve the imaging of intact spheroids by CLSM. Furthermore, this work may also contribute to increase the application of 3-D cell culture models by the pharmaceutical industry for the high-throughput screening of therapeutics.

Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display.

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

A novel method for oxygen glucose deprivation model in organotypic spinal cord slices.

This study aimed to establish a model to closely mimic spinal cord hypoxic-ischemic injury with high production and high reproducibility. Fourteen-day cultured organotypic spinal cord slices were divided into 4 groups: control (Ctrl), oxygen glucose deprived for 30min (OGD 30min), OGD 60min, and OGD 120min. The Ctrl slices were incubated with 1ml propidium iodide (PI) solution (5μg/ml) for 30min. The OGD groups were incubated with 1ml glucose-free DMEM/F12 medium and 5μl PI solution (1mg/ml) for 30min, 60min and 120min, respectively. Positive control slice was fixed by 4% paraformaldehyde for 20min. The culture medium in each group was then collected and the Lactate Dehydrogenase (LDH) level in the medium was tested using Multi-Analyte ELISArray kits. Structure and refraction of the spinal cord slices were observed by light microscope. Fluorescence intensity of PI was examined by fluorescence microscopy and was tested by IPP Software. Morphology of astrocytes was observed by immunofluorescence histochemistry. Caspase 3 and caspase 3 active in different groups were tested by Western blot. In the OGD groups, the refraction of spinal cord slices decreased and the structure was unclear. The changes of refraction and structure in the OGD 120min group were similar to that in the positive control slice. Astrocyte morphology changed significantly. With the increase of OGD time, processes became thick and twisted, and nuclear condensations became more apparent. Obvious changes in morphology were observed in the OGD 60min group, and normal morphology disappeared in the OGD 120min group. Fluorescence intensity of PI increased along with the extension of OGD time. The difference was significant between 30min and 60min, but not significant between 60min and 120min. The intensity at OGD 120min was close to that in the positive control. Compare with the Ctrl group, the OGD groups had significantly higher LDH levels and caspase 3 active/caspase 3 ratios. The values increased with the extension of OGD time and reached peak at 120min. The increase was significant between 30min and 60min, but not significant between 60min and 120min. Organotypic spinal cord slices cultured in glucose-free medium and anaerobic incubator could mimic hypoxia-ischemia of the spinal cord perfectly; 60min could be the best duration for OGD. This technique might be a simple and efficient method to obtain in vitro model for spinal cord hypoxic-ischemic injury in sufficient number and with high quality.

Effects of Gualou Guizhi Decoction Aqueous Extract on Axonal Regeneration in Organotypic Cortical Slice Culture after Oxygen-Glucose Deprivation.

Gualou Guizhi decoction (GLGZD) is effective for the clinical treatment of limb spasms caused by ischemic stroke, but its underlying mechanism is unclear. Propidium iodide (PI) fluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), immunohistochemistry, western blot, and real-time qPCR were used to observe the axonal regeneration and neuroprotective effects of GLGZD aqueous extract on organotypic cortical slices exposed to oxygen-glucose deprivation (OGD) and further elucidate the potential mechanisms. Compared with the OGD group, the GLGZD aqueous extract decreased the red PI fluorescence intensity; inhibited neuronal apoptosis; improved the growth of slice axons; upregulated the protein expression of tau and growth-associated protein-43; and decreased protein and mRNA expression of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor 1 (NgR1), ras homolog gene family A (RhoA), rho-associated coiled-coil-containing protein kinase (ROCK), and phosphorylation of collapsin response mediator protein 2 (CRMP2). Our study found that GLGZD had a strong neuroprotective effect on brain slices after OGD injury. GLGZD plays a vital role in promoting axonal remodeling and functional remodeling, which may be related to regulation of the expression of Nogo-A and its receptor NgR1, near the injured axons, inhibition of the Rho-ROCK pathway, and reduction of CRMP2 phosphorylation.

Iron-induced necrotic brain cell death in rats with different aerobic capacity.

Brain iron overload has a key role in brain injury after intracerebral hemorrhage (ICH). Our recent study demonstrated that ICH-induced brain injury was greater in low capacity runner (LCR) than in high capacity runner (HCR) rats. The present study examines whether iron-induced brain injury differs between LCRs and HCRs. Adult male LCR and HCR rats had an intracaudate injection of iron or saline. Rats were euthanized at 2 and at 24 h after T2 magnetic resonance imaging, and the brains were used for immunostaining and Western blotting. LCRs had more hemispheric swelling, T2 lesion volumes, blood-brain barrier disruption, and neuronal death at 24 h after iron injection (p < 0.05). Many propidium iodide (PI)-positive cells, indicative of necrotic cell death, were observed in the ipsilateral basal ganglia of both HCRs and LCRs at 2 h after iron injection. PI fluorescence intensity was higher in LCRs than in HCRs. In addition, membrane attack complex (MAC) expression was increased at 2 h after iron injection and was higher in LCRs than in HCRs. The PI-positive cells co-localized with MAC-positive cells in the ipsilateral basal ganglia. Iron induces more severe necrotic brain cell death, brain swelling, and blood-brain barrier disruption in LCR rats, which may be related with complement activation and MAC formation.

Detection of live/dead Staphylococcus aureus cells based on CdSe quantum dots and propidium iodide fluorescent labeling

A method of detecting live/dead Staphylococcus aureus cells was developed based on CdSe quantum dots-immunoglobulin G and propidium iodide fluorescent labeling. CdSe quantum dots were synthesized and surface-modified with immunoglobulin G and subsequently subjected to label S. aureus. The live S. aureus cells were stained by CdSe QDs-IgG using S. aureus protein A as target. The results showed that CdSe quantum dots with carboxyl were highly luminescent, stable, and successfully conjugated with the immunoglobulin G after staining (40 min). The optimal concentration of the immunoglobulin G coupled with CdSe quantum dots (1.0 mg/ml) was 80 ng/ml. Quantum dots-immunoglobulin G had high affinity to the surface protein A of S. aureus and exhibited high recognition property for three pathogenic S. aureus compared to Escherichia coli,Streptococcus thermophilus, and one non-pathogenic S. aureus. The fluorescence intensity of CdSe quantum dots decreased with an increasing ratio of dead S. aureus, while the fluorescence intensity of propidium iodide increased. Key words: Staphylococcus aureus, CdSe quantum dots, Staphylococcus aureusprotein A, immunoglobulin G, propidium iodide, fuorescence resonance energy transfer.

Continuous spectroscopic measurements of photo-stimulated release of molecules by nanomachines in a single living cell

The first continuous, real-time spectroscopic monitoring of a photo-driven cargo delivery event from a mesoporous silica-based nanocarrier inside a single living cell is reported. By chemically attaching azobenzene molecules inside the 3 nm pore channels of mesoporous silica nanoparticles (∼70 nm diameter), the escape of the cargo molecule [propidium iodide (PI)] from the pore is prevented in the dark but is facilitated by the light-driven isomerization motion. Real-time spectroscopic measurements of a single cell uncover intermediate processes that occur during this intracellular delivery event, from nanomachine activation to the release of PI into the cytosol and to PI's eventual intercalation with nuclear DNA. Changes in PI's fluorescence intensity and the hypsochromic shift of the band maxima are used to identify the local environment of the fluorophore that is being observed in the cell. The ability to precisely initiate a chemical event inside an individual cell and continuously monitor the subsequent biological responses will enhance our understanding of intracellular process upon drug, protein and nucleic acid delivery.

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1. Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11. Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population. A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements(1). Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations(2). Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer(3). The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis(4-6) and cell viability analysis with propidium iodide (PI)(7-11). Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population. A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

DIDS protects against neuronal injury by blocking Toll‐like receptor 2 activated‐mechanisms

Using an in vitro ischemia model (ischemic solution; IS model) that induces penumbral cell death, we examined the effect of 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS) on cell injury/death and underlying molecular mechanisms. Propidium iodide (PI) uptake was used to quantify cell death in organotypic hippocampal slice cultures. A 24-h IS exposure caused a fivefold increase in mean PI fluorescence intensity. DIDS, dose-dependently (1-4000 microM), reduced the IS-induced PI uptake in hippocampal CA1 neurons with an IC(50) of 26 microM. This protective effect of DIDS was reversible and effective even 6 h following the onset of IS treatment. Gene expression profiling studies indicated that among approximately 46,000 transcripts tested, the most significantly up-regulated gene by IS was interleukin-1beta (IL-1beta) which was also the most significantly down-regulated gene when DIDS was added to the IS-treated slices. The addition of a recombinant IL-1 receptor antagonist (100 microg/mL) or neutralizing IL-1beta antibody significantly attenuated the IS-induced cell death, indicating that the up-regulation of IL-1beta with IS treatment contributed to the IS-induced cell death. Toll-like receptor 2 (TLR2), another gene that was significantly up-regulated by IS and suppressed by DIDS, was studied to determine whether it was related to the IL-1beta up-regulation. Indeed, this was the case as the IS-induced IL-1beta up-regulation was abolished in TLR2-/- mouse brain slices. Furthermore, the IS-induced cell death was significantly reduced in TLR2-/- when compared with that in wild-type slices, indicating that TLR2 is functionally upstream of IL-1beta in this IS model. We conclude that (i) IS up-regulates TLR2 expression and augments TLR2 signaling, causing over-expression of IL-1beta which leads to cell death and (ii) DIDS blocks IS-induced neuronal injury, at least partially, by suppressing the TLR2 pathway.

Acute neuronal injury after hypoxia is influenced by the reoxygenation mode in juvenile hippocampal slice cultures

In neonates asphyxia is usually followed by hyperoxia due to resuscitation procedures. In order to study whether hyperoxic reoxygenation might cause additional cell injury we subjected organotypic hippocampal slice cultures of juvenile rats to normoxic or hyperoxic reoxygenation (19 or 85% oxygen, respectively) following hypoxia (3% oxygen) for 30, 60, and 120 min. Cell injury was quantified by lactate dehydrogenase (LDH) release and propidium iodide (PI) fluorescence 1 h after end of the reoxygenation period. In both experimental groups, LDH activity was significantly enhanced by hypoxia as compared to normoxic controls. However, hyperoxic reoxygenation caused a larger increase in LDH activity than normoxic reoxygenation (e.g., by a factor of 1.60 vs. 1.29 following 120 min hypoxia). PI fluorescence increased after hypoxia in all principal cell layers of the hippocampus but again showed a larger enhancement after hyperoxic reoxygenation as compared to normoxic reoxygenation (e.g., by a factor of 3.9 vs. 1.7 for CA1 and 120 min of hypoxia). After normoxic reoxygenation, PI fluorescence intensity was lower in the dentate gyrus as compared to CA1 and CA3, while it reached similar values like CA1 after high oxygen supply. In conclusion, juvenile hippocampal slice cultures subjected to hyperoxic reoxygenation display a greater amount of acute neuronal injury than slice cultures undergoing normoxic reoxygenation.

A nitroimidazole derivative, PR-350, enhances the killing of pancreatic cancer cells exposed to high-dose irradiation under hypoxia.

The radiosensitizing effects of PR-350, a nitroimidazole derivative, were examined concerning the cell killing of human pancreatic cancer cell lines exposed to high doses of gamma-ray irradiation in vitro. The percentages of dead cells were analyzed with a multiwell plate reader to measure the fluorescence intensity of propidium iodide before and after a digitonin treatment. The sensitizing effect of PR-350 on cell killing by high-dose irradiation was confirmed by time-course, dose-dependency, and microscopic observations. In five of seven pancreatic cancer cell lines in which the number of dead cells was determined 5 days after 30 Gy irradiation in the presence of PR-350, the number was significantly increased under hypoxic conditions, but not under aerobic conditions. The selective radiosensitive effect of PR-350 on hypoxic cells was also confirmed by flow cytometry. The results indicate that PR-350 can enhance the killing of pancreatic cancer cells by high-dose irradiation under hypoxia, which supports its clinical radiosensitizing effects when administered during intraoperative irradiation to pancreatic cancer.

The photosensitizing effect of the photoproduct of protoporphyrin IX

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm 2 light at 635 nm, the photoproduct — the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter.

The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.

Glutamate-mediated neuroprotection against N-methyl- d-aspartate toxicity: a role for metabotropic glutamate receptors

We studied N-methyl- d-aspartate-induced cell death in organotypic hippocampal slices from seven-day-old Wistar rat pups cultured for 12–14 days in a medium containing no added glutamate. Propidium iodide fluorescence intensity was used as an indicator of cell death measured with the help of confocal microscopy. Exposure of slices for 2 h to l-glutamate (1–500 μM) prior to the N-methyl- d-aspartate challenge significantly reduced N-methyl- d-aspartate-induced cell death. Glutamate at 10 and 500 μM concentrations was highly protective against N-methyl- d-aspartate-induced cell death, but was less protective at the 1 μM concentration. The protection was not blocked by the Na + channel blocker tetrodotoxin (1 μM), the N-methyl- d-aspartate receptor antagonist d-2-amino-5-phosphonopentanoic acid (20 μM) or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM). 1 S,3 R-1-Aminocyclopentane- trans-1,3-dicarboxylic acid, an agonist at metabotropic glutamate receptor types 1, 2/3 and 5, was protective at 100 μM but not at 50 μM. In contrast, the ionotropic glutamate receptor agonist aspartate (250 μM) facilitated N-methyl- d-aspartate toxicity. Treatment of slices with the protein kinase C inhibitor staurosporine (0.2 μM) or antisense oligonucleotide (10 nM, 72 h) that selectively inhibits metabotropic glutamate receptor type 5 synthesis significantly reduced glutamate protection. These results suggest that ambient glutamate may reduce nerve cell susceptibility to injury caused by excessive N-methyl- d-aspartate receptor activation by acting at metabotropic glutamate receptors linked to protein kinase C.