Light-scattering Changes Research Articles

Supravital exposure to propidium iodide identifies apoptosis on adherent cells

Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

A portable, non-focusing optics spectrophotometer (NoFOSpec) for measurements of steady-state absorbance changes in intact plants.

Kinetically-resolved absorbance measurements during extended, or steady-state illumination are typically hindered by large, light-induced changes in the light-scattering properties of the material. In this work, a new type of portable spectrophotometer, the Non-Focusing Optical Spectrophotometer (NoFOSpec), is introduced, which reduces interference from light-scattering changes and is in a form suitable for fieldwork. The instrument employs a non-focusing optical component, called a compound parabolic concentrator (CPC), to simultaneously concentrate and homogeneously diffuse measuring and actinic light (from light-emitting diode sources) onto the leaf sample. Light passing through the sample is then collected and processed using a subsequent series of CPCs leading to a photodiode detector. The instrument is designed to be compact, lightweight and rugged for field work. The pulsed measuring beam allows for high sensitivity (typically < 100 ppm noise) and time resolution ( approximately 10 mus) measurements in the visible and near infrared spectral regions. These attributes allow high-resolution measurements of signals associated with energization of the thylakoid membrane (the electrochromic shifting of carotenoid pigments), as well as electron transfer, e.g., the 820-nm changes associated with electron transfer through Photosystem I (PS I). In addition, the instrument can be used as a kinetic fluorimeter, e.g., to measure saturation-pulse fluorescence changes indicative of Photosystem II (PS II) quantum efficiency. The instrument is demonstrated by estimating electron and proton fluxes through the photosynthetic apparatus in an intact tobacco leaf, using respectively the saturation-pulse fluorescence changes and dark-interval relaxation kinetics (DIRK) of the electrochromic shift. A linear relationship was found, confirming our earlier results with the laboratory-based diffused-optics flash spectrophotometer, indicating a constant H(+)/e(-) stoichiometry for linear electron transfer, and suggesting that cyclic electron flow around PS I is either negligible or proportional to linear electron flow. This type of measurement should be useful under field conditions for estimating the extent of PS I cyclic electron transfer, which is proposed to operate under stressed conditions.

Morphological Differences between GPIb Antibody-Induced and Shear Stress-Induced Platelet Aggregates

We determined the morphological differences between NNKY5-5-induced and shear stress-induced platelet aggregates using confocal laser scanning microscopy, and investigated the effects of cytokines on these aggregates. As shear stress was increased from low to high, many aggregates were generated. Some platelets and aggregates already exhibited PAC-1 binding at low shear stress. And finally, aggregates were clearly stained by PAC-1 at high shear stress. Low shear stress-induced aggregates included fibrinogen, but not von Willebrand factor (vWF). In contrast, high shear stress-induced aggregates included both fibrinogen and vWF. NNKY5-5-induced platelet aggregates exhibited both PAC-1 binding and fibrinogen, but vWF was not found in them. The aggregate formation in NNKY5-5-treated platelet-rich plasma (PRP) at low shear stress was clearly more pronounced than that in untreated PRP. In addition, aggregates in NNKY5-5-treated PRP formed after 6 min under low shear stress were clearly stained by PAC-1 and included fibrinogen, but vWF was not found in them. In order to investigate the effects of cytokines on platelet activation under NNKY5-5 stimulation, changes in light transmission and light scatter were assessed with an AG-10. G-CSF, IL-6 and thrombopoietin (TPO) each enhanced light scatter compared to control PRP, although IL-3, GM-CSF, erythropoietin and stem cell factor did not. In particular, TPO significantly enhanced the ‘%-T’ and ‘S-Max’ of NNKY5-5-treated PRP. TPO clearly enhanced the aggregate formation with high shear stress or NNKY5-5 stimulation. These results suggest that the glycoprotein Ib (GPIb)-unmediated aggregates can be formed with only fibrinogen, but the GPIb-mediated aggregates by vWF and fibrinogen synergistically. In particular, the latter is formed more firmly, and both aggregates are enhanced by TPO.

A rapid measurement of apoptosis‐associated light scatter changes using a hematology analyzer

A rapid measurement of apoptosis‐associated light scatter changes using a hematology analyzer

Automated quantitation of circulating neutrophil and eosinophil activation in asthmatic patients

BACKGROUNDAsthma has been associated with eosinophil activation, measured in serum, sputum, bronchoalveolar lavage (BAL) fluid, and urine. A whole blood automated method was developed to assess eosinophil and neutrophil activity...

A rapid measurement of apoptosis-associated light scatter changes using a hematology analyzer

The alternative application of an automated hematology analyzer, H*3 system, has been described for the detection of apoptosis. Apoptosis induction by the topoisomerase I inhibitor, camptothecin (CAM) on several cell lines is followed by typical morphological alterations. On the H*3 cytogram, measurement of CAM-treated cells revealed an increased population of cells with reduced size suggesting cell contraction during apoptosis. The decreased LUC/Lymph ratio also indicated the enhanced degree of apoptosis directly correlated with increasing CAM concentration and/or incubation period. Quantitative analysis shows a good correlation between the H*3 measurement and flow cytometry measurements of Annexin V-fluorescein isothiocyanate-labeled method. Thus, the H*3 measurement, under an appropriate adjustment, can be used as a rapid monitor for evaluating the degree of apoptotic changes in drug susceptibility testing of homogeneous cell samples.

APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model.

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.

Quantitative automated particle-enhanced immunonephelometric assay for the routinary measurement of human cystatin C.

Human cystatin C is a low molecular mass protein of 13359 Dalton recently proposed as a new very sensitive marker of changes in glomerular filtration rate. Serum cystatin C concentration correlates negatively with glomerular filtration rate as well as or better than creatinine. We evaluated a recently introduced automated nephelometric immunoassay for cystatin C in serum or EDTA-plasma samples on the Behring Nephelometer System. The assay consists of incubating the 100-fold diluted sample for 6 minutes with latex particles covalently coated with anti-human cystatin C antibodies, and then quantifying the change of light-scatter produced. Method reproducibility is satisfactory, the intra- and inter-assay coefficients of variation ranging from 1.58% to 3.77% and from 5.6% to 11.47% respectively. Rheumatoid factor (< or = 1116 IU/ml), bilirubin (< or = 418 micrommol/l), triglycerides (10.47 mmol/), and haemoglobin (12 g/l) do not significantly interfere in the assay. No significant difference was found in cystatin C concentration between serum and EDTA-plasma samples. Cystatin C is stable in serum samples stored under different conditions up to one month. This method correlates well (mean difference=-0.536+/-0.307 mg/l) with another commercially available particle-enhanced turbidimetric immunoassay. Cystatin C offers better clinical sensitivity than creatinine for discriminating patients with normal renal function and those with mild-to-moderate reduction in renal function. This method is suitable for routine cystatin C measurement, including emergencies.

Optimized fixation and storage conditions for FISH analysis of single-cell suspensions.

In our protocol to isolate and identify fetal cells in maternal peripheral blood, antibody (Ab)-stained cells are preserved with paraformaldehyde (PF) before batch flow cytometric sorting. However, PF fixation compromises the quality of subsequent interphase fluorescence in situ hybridization (FISH). We therefore examined the effect of PF concentrations and storage time in phosphate-buffered saline (PBS) on the quality of FISH signals. Cells were analyzed for changes in light scatter, morphology, and accessibility of target cell DNA. Fixation in 3% PF for 1 hr was ideal for both flow cytometry and subsequent FISH detection. However, beyond 10 days of storage, FISH quality deteriorated. (J Histochem Cytochem 46:971-973, 1998)

Measurement ofCandida-specific blastogenesis: Comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression

Measurement of the T cell blastogenic response to Candida may be useful in the evaluation of patients with suspected immunodeficiency. The classic blastogenesis assay is based on uptake of [3H]thymidine by peripheral blood lymphocytes stimulated with Candida antigens for 5 days. An alternative approach involves staining peripheral blood lymphocytes with the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and measuring mitotic activity by the successive twofold reductions in fluorescent intensity using flow cytometry (FCM). The two approaches were compared in 16 subjects who demonstrated various proliferative responses to Candida. FCM-derived indices all involved initial gating on CD3+ T cells and included 1) blastic transformation as measured by changes in light scatter, 2) cell division, measured by CFSE fluorescence, and 3) CD69 expression. A good correlation was found between [3H]thymidine uptake and CFSE-derived indices, irrespective of the analysis algorithm used to interpret CFSE division profiles. Furthermore, significant T cell proliferation occurred only in subjects who had had one or more symptomatic episodes of vaginal candidiasis whereas controls with no such history, and patients with chronic vaginal infection, showed minimal proliferation. The increase in proportion of CD69+ T cells in culture also correlated with the blastogenic response to Candida, but less well than mitotic indices. CFSE-derived indices of T cell blastogenesis to Candida are equivalent to [3H]thymidine-based assays and may allow useful laboratory distinction between subjects who have been exposed to and recovered from vaginal Candida infection, who have a strong proliferative response, from those with no exposure or chronic infection who demonstrate a poor response.

GABA-Induced intrinsic light-scattering changes associated with voltage-sensitive dye signals in embryonic brain stem slices: coupling of depolarization and cell shrinkage.

We have found new evidence for gamma-aminobutyric acid (GABA)-induced intrinsic optical changes associated with a voltage-sensitive dye signal in the early embryonic chick brain stem slice. The slices were prepared from 8-day-old embryos, and they were stained with a voltage-sensitive dye (NK2761). Pressure ejection of GABA to one site within the preparation elicited optical changes. With 580-nm incident light, two components were identified in the GABA-induced optical change. The first component was wavelength dependent, whereas the second, slower change was independent of wavelength. Comparison with the known action spectrum of the dye indicates that the first component reflects a depolarization of the membrane and that the second, slow component is a light-scattering change resulting from cell shrinkage coupled with the depolarization. Similar optical changes also were induced by glycine, although the amplitude of both the first and second signals was much smaller than for GABA. The optical changes induced by GABA persisted in the presence of picrotoxin and 2-hydroxysaclofen, suggesting that these optical responses include a novel GABA response, which has been termed GABAD in our previous reports.

A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.

Intrinsic light-scattering changes coupled with glutamate-induced membrane depolarization in embryonic brainstem slices

Ultrasound has evolved in dermatology from an experimental phase to a daily practice imaging technique. Its several advantages include its safety, good balance between penetration and resolution, high definition, and the detection of blood flow in real time. Its applications are growing and include the support of the diagnosis and extent in all axes, including depth, vascularity patterns, staging, and follow up of multiple cutaneous diseases—benign cutaneous tumors, vascular anomalies, nail lesions, skin cancer, inflammatory cutaneous diseases, and aesthetics complications.

Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein-specific monoclonal antibody APO2.7

A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.

Optical illustration of glutamate-induced cell swelling coupled with membrane depolarization in embryonic brain stem slices.

Using intrinsic- and voltage-sensitive dye optical recordings, we have elucidated coupling of glutamate-induced depolarization and neuronal swelling in early embryonic chick brain stem slices. Twenty-four slices were prepared from 8-day old chick embryos, and stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). The pressure ejection of glutamate to one site within the preparation evoked changes in transmitted light intensity. With 700 nm incident light, three components were identified in glutamate-induced optical changes. The first component was wavelength dependent, while the second and third components were independent of the wavelength. With reference to the action spectrum of the merocyanine-rhodanine dye and osmotic changes in optical properties, we concluded that the first component reflects glutamate-induced depolarization of the membrane, and that the second component is an intrinsic light-scattering change resulting from neural cell swelling coupled with the membrane depolarization.

The use of Apostain in identifying early apoptosis

Irradiated human peripheral blood lymphoid cells undergo apoptosis and progressively exhibit typical changes in light scatter and plasma membrane integrity that can be easily tracked by flow cytometry. Using this model, we assessed the capacity of a newly developed fluorochrome, Apostain, in identifying early apoptosis in unfixed samples. This probe is a plasma membrane permeant DNA dye that can be conveniently excited at 488 nm and has an emission wavelength >650 nm. To identify apoptotic cells, Apostain relies on the transient changes of chromatin texture that allow to accommodate more of a DNA dye occurring in early apoptosis. As early as 4 h after irradiation, a proportion of cells showed an enhanced Apostain uptake. Consistent with their initial apoptotic nature, these cells had a still integer plasma membrane, as assessed by ethidium bromide, and unaltered light scatter. With time, cells showing the enhanced Apostain uptake started to bind dimly Annexin-V and, later, reduced their forward scatter. After 18 h from irradiation, cells exhibiting a reduced forward scatter exhibited a bright staining with Annexin-V with a concomitant reduction in Apostain uptake, reflecting the gross chromatin disruption characterising the endpoint of apoptosis.

Osmotic and Metabolic‐Induced Changes in Light Scattering of Leishmania donovani as Measured by Flow Cytometry

Leishmania donovani promastigotes were collected from cultures in log and stationary phases of growth and resuspended in Hank's Balanced Salt Solution containing 1 mM sodium acetate. Changes in the forward and side scattering of the cells were measured by flow cytometry in response to acute changes in osmolality and to the addition of several different substrates. Forward and side scattering of cells from log phase cultures decreased when the osmolality was decreased by the addition of H2O and increased when the osmolality was increased by the addition of NaCl. Cells from stationary phase cultures had about the same forward scatter as cells from log phase cultures, but almost a four‐fold lower side scatter, and their side scatter values did not change significantly in response to a reduction in osmolality. Microscopic observation showed that both log and stationary cells got longer and thinner, on average, in response to hyperosmolality. The light scattering properties of log (but not of stationary) cells changed in a reproducible manner when substrates were added to the buffer. The ratio of forward to side scatter increased in the following order: controls in balanced salt solution &gt;aspartate &gt;glutamate, glucose or 2‐deoxyglucose &gt;alanine &gt;proline. Thus the light scattering properties of L. donovani promastigotes change with culture age, in response to changes in osmolality, and, in log phase cells, in response to the presence of several substrates.

Gadolinium induces macrophage apoptosis

Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3.6H20 caused cell death, as measured by trypan blue permeability, in both dose- and time-dependent fashions. Even a 10-min exposure to Gd caused significant cell death by 24 h. The morphology of Gd-treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd-treated propidium iodide-excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd-treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron-dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

Rapid Changes in Light-Scattering in the Prism ofTorpedoElectric Organ Slice Associated with the Production of Postsynaptic Potentials

Using a bifurcated lightguide placed on the surface of a prism of the electric organ slice ofTorpedo,the generation of postsynaptic potentials by direct stimulation was found to be associated with readily detectable, disphasic optical changes. The changes consisted of a decrease in light-scattering during the rising phase of the electric potential, followed by a prolonged increase in scattering. The effects of varying the calcium-ion concentration in the medium strongly support the view that replacement of transmitter molecules for bound Ca-ions in the receptor proteins is at the base of these changes. The effects of synaptically active reagents on these optical signals were demonstrated.

Visual efficiency of scattering and fluorescence in the human eye lens

Recent data in the literature on psychophysically determined in vivo intraocular straylight, and on in vitro fluorescence of the eye lens are analyzed. From the psychophysical straylight data, light scattering changes in the lens due to normal aging and age-related cataract formation are derived in physical terms. The intensities of these light-scattering changes prove to follow approximately cu p (u=scatter angle) with p'−2 and c dependent on age and cataract. Both p and c are in accordance with recent in vitro studies on light scattering using donor lenses. Fluorescence of the lens causes light with wavelengths of 420 nm and lower to be in total visually much more effective: by a factor of 2.7 to 6.8 at 400 nm, and a factor of 71 to 151 at 380 nm. Because fluorescence adds a homogeneous veil to the point spread function, for some visual effects (e.g., glare) the increase can be (much) larger.