Cytometry最新文献

Background: The objective of this study was to characterize and quantitate the calcium responses to cholinergic stimulation in individual primary rat cortical astrocytes and human 132 1N1 astrocytoma cells. Materials and Methods The fluorescent calcium probe Indo-1 AM and an attached cell analysis and sorting (ACAS) instrument were used to quantitate calcium responses in these cells.

Results: A concentration-dependent response to carbachol was seen in both cell types. However, carbachol was more potent and efficacious, and the response was more homogeneous in the cell line. The calcium response was mediated by the M3 subtype of muscarinic receptors. Experiments in the absence of extracellular calcium and with EGTA demonstrated that the initial calcium spike was due to calcium release from intracellular calcium stores, whereas the sustained elevation and oscillations were dependent on calcium influx. Protein kinase C exerts a feedback inhibition of these calcium responses, and appears to be involved in maintaining the elevated calcium concentration and oscillations.

Conclusions: This study provided a detailed quantitation of the changes in intracellular calcium evoked in individual astroglial cells by activation of M3 muscarinic receptors. This will allow for the study of pharmacological and toxicological agents on this response.

Background: The discriminatory power and imaging efficiency of different multicolor FISH (M-FISH) analysis systems are key factors in obtaining accurate and reproducible classification results. In a recent paper, Garini et al. put forth an analytical technique to quantify the discriminatory power ("S/N ratio") and imaging efficiency ('excitation efficiency') of multicolor fluorescent karyotyping systems.

Methods: A parametric model of multicolor fluorescence microscopy, based on the Beer-Lambert law, is analyzed and reduced to a simple expression for S/N ratio. Parameters for individual system configurations are then plugged into the model for comparison purposes.

Results: We found that several invalid assumptions, which are used to reduce the complex mathematics of the Beer-Lambert law to a simple S/N ratio, result in some completely misleading conclusions about classification accuracy. The authors omit the most significant noise source, and consider only one highly abstract and unrepresentative situation. Unwisely chosen parameters used in the examples lead to predictions that are not consistent with actual results.

Conclusions: The earlier paper presents an inaccurate view of the M-FISH situation. In this short communication, we point out several inaccurate assumptions in the mathematical development of Garini et al. and the poor choices of parameters in their examples. We show results obtained with different imaging systems that indicate that reliable and comparable results are obtained if the metaphase samples are well-hybridized. We also conclude that so-called biochemical noise, not photon noise, is the primary factor that limits pixel classification accuracy, given reasonable exposure times.

Background: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry.

Results: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5.

Conclusions: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.

Background: We have shown, in a rat model of immunodeficiency, permanent alterations in the thymus and in the gut-associated lymphoid tissues. We observed by immunohistochemistry an increase in the number of gamma/delta+ T cells in the gut lamina propria and in the number of CD8alpha/alpha+, CD25+, gamma/delta+ subpopulations of intestinal intraepithelial lymphocytes (iIEL). The aim of the present study was to analyze the isolated rat iIEL by flow cytometry. Materials and Methods Cells from mesenteric lymph nodes were examined in parallel with isolated iIEL. After staining with different antibodies, samples were run on a FACScan flow cytometer. Background staining was evaluated using isotype controls. Data analysis was performed using Lysys II software (Becton Dickinson) and WinMDI 2.3 software.

Results: 1) CD8alpha/beta populations do not express TCRgamma/delta, 2) CD8alpha/alpha+ populations express TCRgamma/delta, and its percentage is significantly increased in R21, 3) CD8alpha/beta and CD8alpha/alpha iIEL express TCRalpha/beta, being the percentage of CD8alpha/alpha+ TCRalpha/beta+ iIEL increased and the percentage of CD8alpha/beta+ TCRalpha/beta+ iIEL decreased in R21, and 4) CD8alpha/alpha as well as CD8alpha/beta iIEL do express CD25 only in R21.

Conclusions: Considering the above results, we conclude that there exists an "in situ" origin and extrathymic maturation of the CD8alpha/alpha+ iIEL in the intestinal epithelium. The increase of TCRgamma/delta+ T cells may be triggered by the carbohydrate dextrin, to provide immune protection and control of inflammation at the intestinal level.

Background: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation.

Methods: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin.

Results: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells.

Conclusions: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.

Background: To identify nuclei and lesions with great specificity, a large set of karyometric features is arranged in the form of a linear profile, called a nuclear signature. The karyometric feature values are normalized as z-values. Their ordering along the profile axis is arbitrary but consistent. The profile of the nuclear signature is distinctive; it can be characterized by a new set of variables called contour features. A number of data reduction methods are introduced and their performance is compared with that of the karyometric features in the classification of prostatic, colonic, and esophageal lesions.

Methods: Contour characteristics were reduced to descriptive statistics of the set of z-values in the nuclear signature and to sequence information. The contour features derived were (1) relative frequencies of occurrence of z-values and of their differences and (2) co-occurrence statistics, run lengths of z-values, and statistics of higher-order dependencies. Performance was evaluated by comparing classification scores of diagnostic groups.

Results: Rates for correct classification by karyometric features alone and contour features alone indicate equivalent performance. Classification by a combined set of features led to an increase in correct classification.

Conclusions: Image analysis and subsequent data reduction of nuclear signatures of contour features is a novel method, providing quantitative information that may lead to an effective identification of nuclei and lesions.

Background: Estimating the duration of S phase (T(S) ) and the potential doubling time (T(pot) ) from a single time measurement of the movement of cells using bivariate cytometry is common. However, these estimates require an assumption of the duration of G2 + M (T(G2+M) ). Inspection of the measured dynamic quantities, relative movement [RM(t)], fractions of labeled divided and undivided cells (f(lu)(t) and f(ld)(t)) suggests that T(G2+M), T(S), and T(pot) can be determined simultaneously.

Methods: An equation connecting the growth of the cell population, time, and the dynamic quantities was determined. The equation cannot be solved analytically, but accurate approximations can be used to find T(pot). From this result, the value of T(G2+M) can be determined from f(ld)(t), and T(S) can be determined from RM(t).

Results: Kinetic parameters obtained from single time estimates using the new method compared to those obtained from the analysis of multiple time-point measurements of MCa-K and MCa-4 murine tumors are shown to be in close agreement. Moreover, estimates of T(G2+M) in MCa-4 tumors, treated with paclitaxel, provide extra information on the changes in T(G2+M). When applied to the rat R3327-G prostate tumor model following androgen ablation, a correlation analysis of the T(pot) values obtained by the new and previous single time-point methods demonstrates that the rank order from shortest to longest T(pot) values are largely preserved.

Conclusions: The new procedure makes direct estimation of T(G2+M) possible from single time-dynamic measurements. The results from previous studies on T(S) and T(pot) are largely unchanged, but extra information is now available.

Background: Many times small differences in genome size are reported between or within plant species in which no cytologic confirmation is made. Attempts to repeat these studies have met with limited success. The controversy then becomes whether or not these small differences that were not confirmed cytologically are real. The present study was undertaken to determine if the approximately 1% nuclear variation seen by flow cytometry among wheat lines selected for aluminum response was due to actual chromatin differences.

Methods: The three parental wheat cultivars used in the aluminum selection along with the isolines resulting from the selection were analyzed. One parental line had previously been reported to have alien chromatin substituted for the corresponding wheat homologous chromatin. Genomic in situ hybridization was used to determine the presence or absence of rye chromatin in three cultivars and six near-isolines of wheat.

Results: Upon observing metaphase chromosomes of the Century parent and its isolines, two of the chromosomes were observed to be one-half yellow-orange, indicating rye chromatin with the remaining portion of the chromosomes and the other 40 wheat chromosomes having no label indicating wheat chromatin. In the Chisholm parent and its isolines, none of the chromosomes were labeled, indicating the absence of rye chromatin. In addition, none of the third parents' chromosomes had the rye yellow-orange signal.

Conclusions: The wheat lines with the larger DNA contents were observed to have alien DNA present. DNA differences between the normal wheat chromosomes and the substituting alien chromatin were calculated based on total chromosome length. The increase in genome size of the wheat lines containing the alien chromatin appears to be the result of the alien chromatin having approximately 43% more DNA than the wheat chromatin it is replacing. Thus, the small DNA difference previously reported by flow cytometry was demonstrated to be a real DNA variation due to the presence of small fragments of alien chromosomes added to the wheat genome.

Background: Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments.

Methods: Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation.

Results: Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane.

Conclusions: We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.