Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.     

Quantification of chemotactic response of quiescent and proliferating fibroblasts in Boyden chambers by computer-assisted image analysis

The chemotactic response of human gingival fibroblasts to platelet-derived growth factor (PDGF) was investigated in 48-well modified Boyden chambers. Results were quantified using computer-assisted image analysis of propidium iodidestained cells and were compared with results obtained using the conventional method of quantification by direct counting. Quantification by image analysis was found to be rapid and accurate, and correlated closely with results obtained by direct counting. Bromodeoxyuridine (BrdU)-labeled proliferating cells were double stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antiserum. The cycling cells showed a markedly reduced chemotactic response to PDGF, whereas cells in S-phase of the cell cycle did not show any response. This method of quantification of results of Boyden chamber assays is simple and reliable, and allows the use of double labels to investigate the chemotactic response of subpopulations of cells within a heterogeneous population of cells.     

Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

Summary Techniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothiocyanate - PI propidium iodide - Tris Tris(hydroxymethyl) aminomethane     

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.     

Flow microfluorometric system for screening gynecologic cytology specimens using propidium iodide-fluorescein isothiocyanate.

Seventy cervical cytology specimens have been screened by a xero resolution flow analyzer-sorter using propidium iodide and fluorescein isothiocyanate as fluorochromes for nucleus and cytoplasm, respectively. This system shows a 1% sensitivity for detection of abnormal cells using only crude visual data analysis. Screening of clinical specimens was performed on the instrument with a 5.8% false negative rate and a 11.8% false positive rate by comparison with routine visual cytologic evaluation of the same samples.     

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 micrograms/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 micrograms lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.     

Flow cytometric analysis of unbalanced control of protein accumulation and DNA synthesis in differentiating mouse myeloid leukemia cells

The protein and DNA contents of mouse myeloid leukemia M1 (clone B24) cells were determined by flow cytometry (FCM) after double fluorescent staining of the cells with fluorescein isothiocyanate and propidium iodide. FCM analysis showed that there was a linear relationship between the DNA and protein contents in logarithmically growing cells, although the protein content showed some variation. B24 cells can be induced to differentiate into macrophage-like cells by treatment with a protein inducer(s) in conditioned medium (CM) of hamster embryo cells. When the cells were treated with various concentrations of CM, cells with a 2C DNA content, G1/0 cells, increased and protein accumulated in these G1/0 cells. The increases in the number of G1/0 cells and in their protein content per cell were proportional to the concentration of CM. Serial analysis of changes in the contents of DNA and protein in differentiating B24 cells showed that DNA synthesis was suppressed by differentiation-induced block of the cell cycle at the G1/0 phase, whereas increase in the protein content was not completely suppressed by block of the cell cycle. These results suggest that unbalanced control of the DNA and protein contents of B24 cells is involved in the mechanisms of the morphological changes during differentiation into macrophages.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Plasmodium falciparum: Automated assay of erythrocyte invasion using flow cytofluorometry

Erythrocytes labeled with fluorescein isothiocyanate, were mixed with erythrocytes infected with Plasmodium falciparum. After allowing time for invasion of labeled cells to take place, cells were stained with propidium iodide. Parasitemia in labeled cells was determined using flow cytofluorometry. The invasion of labeled erythrocytes was reduced in a dose-dependent manner by immune serum. The degree of inhibition obtained increased as the erythrocyte concentration decreased.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

The use of confocal scanning laser microscopy and other tools to characterize Escherichia coli in a high-cell-density synthetic biofilm

Properties of a novel, synthetic biofilm were examined by using confocal scanning laser microscopy (CSLM) in combination with fluorescent probes and by investigating total protein content and specific beta-galactosidase activity during various steps of the biofilm preparation. Viable, but nongrowing Escherichia coli were entrapped in 10- to 80-mum-thick multilayer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. Cell viability and distribution within the films were evaluated by developing a protocol to stain the bacteria with fluorescein isothiocyanate and propidium iodide, thereby labeling all cells green and dead cells red, respectively. Confocal microscopy facilitated viewing samples in the XY and XZ planes, and image analysis enabled counting of the cells. These experiments showed that the initial viability of the entrapped bacteria was 85% to 90%, cell distribution was uniform in the XY plane and cell number increased with increasing depth into the film. Specific beta-galactosidase assays developed here allowed comparison of the induction of lacZin suspended and immobilized cells. These experiments demonstrated that rehydration was an important step in biofilm preparation, and E. coli cast into synthetic biofilms with cell layers of at least 20 to 35 mum in thickness had gene induction characteristics similar to suspended cells. (c) 1996 John Wiley & Sons, Inc.     

The effects of photosensitization by hematoporphyrin derivative on the protein content of cultured human lung cancer cells. A flow cytometric analysis

In order to study the cytotoxic mechanisms of photodynamic therapy (PDT), the changes in the intracellular content of protein and DNA of cultured human lung cancer cells were examined by flow cytometry using the fluorescent dyes fluorescein isothiocyanate (FITC) and propidium iodide (PI). Immediately after PDT, the protein content increased while the DNA content showed little change. After more than 24 hours, the protein and DNA content were markedly decreased in lethally damaged cells; in sublethally damaged cells, however, the protein content gradually decreased to the level of that before PDT while the DNA content showed little change. These changes in the protein content may reflect the impairment of the plasma membrane function by PDT and its subsequent recovery in some cells.     

细胞凋亡调控因子基因在食管上皮癌变过程中的表达及其意义

目的研究细胞凋亡调控因子Fas、Fasl、Fadd和Caspase 8基因在食管上皮癌变过程中的表达及其意义。方法应用免疫组化S-P法检测食管黏膜中Fas、Fasl、Fadd和Caspase 8的蛋白表达。应用碘化丙啶染色和间接免疫荧光标记方法,采用流式细胞仪对食管黏膜组织进行定量检测。结果从食管正常黏膜组织到不典型增生组织和食管癌组织,Fas、Fadd和Caspase 8蛋白表达率呈逐渐下降的趋势,Fasl蛋白表达率呈逐渐上升的趋势,在食管癌和正常组织之间差异有统计学意义,x^2分别为5．659,7．73,6．32,5．65,P〈0．05或P〈0．01。Fas、Fasl和Caspase 8蛋白高中分化鳞癌中蛋白阳性表达率显著高于低分化鳞癌,两者差异有统计学意义,x^2分别为4．88,7．79,4．68,P〈0．05。与正常黏膜组织和不典型增生组织相比,癌组织中DNA含量明显增高,异倍体细胞显著增加;Fas、Fadd和Caspase 8蛋白表达水平明显降低,t值分别为8．131,7．39,5．44,Fasl基因蛋白表达水平升高,t值为6．671。结论Fas与Fasl的表达失衡与食管癌的发生、发展有密切关系,并与食管癌的分化和免疫逃避有关。Fadd与Caspase8基因表达异常在食管癌的发生发展中可能起着重要作用。食管上皮癌变过程中,DNA含量及异倍体率增加。     

Cell-cycle, protein content, and nuclear size in acute myeloid leukemia

Simultaneous analysis of DNA and cellular proteins provides information on cell proliferation and metabolism. Cellular protein content coupled with nuclear geometric parameters can be used to evaluate cellular maturation and differentiation. In this study, leucoblasts from 50 cases of adult acute myeloid leukemia were analyzed by flow cytometry, and semiautomatic morphometry was performed on bone marrow smears. Ethanol-fixed bone marrow blast cells were stained for DNA with propidium iodide (PI) and for proteins with fluorescein isothiocyanate (FITC). On the resulting FITC versus PI histograms we defined the cells with low protein content which are associated with a nonproliferating subpopulation (LPC fraction). Low protein content fraction and S-phase are correlated (p less than 0.01). The LPC fraction values are more dispersed than S-phase values and thus should indicate more clearly eventual differences between cellular populations. This hypothesis has been tested with the prognostic significance of cell-cycle variables: The LPC fraction was significantly higher in the complete remission group than in the other (p less than 0.01), while S-phase did not show any difference. The peak value of the protein content histograms is significantly lower in the granulocytic leukemias (M1, M2, M3) than in the leukemias with a monoblastic component (M4, M5). Furthermore, we showed that the differentiation and the maturation of the myeloid blast cells modify the nuclear size. The combination of these two parameters provides useful information for cytological classification.     

Dual-parameter flow cytometric analysis coupling the measurements of forward-angle light scatter and DNA content of archival ovarian carcinomas of low malignant potential

Paraffin-embedded archival specimens from 45 cases of ovarian carcinoma of low malignant potential (OCLMP) were analyzed by flow cytometry (FCM) using propidium iodide (PI) staining. Since single-parameter FCM analysis is often deficient in the resolution of subtle near-diploid DNA-aneuploid populations, forward-angle light scatter (FALS) was measured as a second parameter. DNA aneuploidy was identified in 15 cases (33%). In 7 of those 15 cases, aneuploidy was resolved with single-parameter FCM; in the remaining 8 cases, DNA aneuploidy was resolved only following dual-parameter analysis coupling DNA content and FALS. In all 15 cases, a single near-diploid aneuploid population was observed (mean DNA index = 1.2); there were no tetraploid aneuploid cases. The proliferative activity for all 45 cases studied ranged from 1.0% to 8.9%, with a mean of 3.5%. No difference in mean proliferative activity was observed between the aneuploid and diploid tumors (P greater than .05). To exclude the possibility that PI staining artifacts caused the observed aneuploidy, five of the eight cases shown to be aneuploid by dual-parameter analysis were further studied using an alternate DNA-binding dye, DAPI, yielding similar results. To exclude the possibility that contaminating stromal and/or inflammatory cells caused the observed aneuploidy, samples from a subset of the dual-parameter cases were sorted, revealing the aneuploid populations to be composed primarily of tumor nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow microfluorometric analysis of cellular DNA: Critical comparison of mithramycin and propidium iodide

Mithramycin and propidium iodide were used to stain HeLa cells, human lymphoma cells, and phytohemagglutinin-stimulated lymphocytes for flow microfluorometric analysis of cellular DNA. The stains provided similar estimates for the proliferative fraction of the populations. However, significant differences in the relative fluorescent intensity were demonstrated in the three cell populations. Fluorescent intensity of HeLa and lymphoma cells stained with mithramycin was higher than matched propidium iodide-stained cells. Normal lymphocytes showed greater fluorescent intensity when stained with propidium iodide. Differences in the staining behavior of these two dyes may prove to be highly informative probes of chromatin structural differences.     

Clinical evaluation for the mechanism of cisplatin with bromodeoxyuridine by flow cytometry and clinico-histological study

The tumor cell kinetics before and after intra-arterial injection of cisplatin (CDDP) was examined in a patient with maxillary carcinoma using flow cytometry and propidium iodide and fluorescein isothiocyanate labeled bromodeoxyuridine double staining. In addition, the viability was determined with fluorescein diacetate and compared with the histological picture. The action mechanism which the authors obtained 24 hours after intra-arterial injection of CDDP to the clinical case was considered to be S phase block, especially early S phase block. Markedly decreased viability also makes us consider the cytocidal effect. These findings correlated with the data of the fundamental investigation reported previously. Histological examination after the intra-arterial injection of CDDP revealed remarkable necrosis in the tissue.     

HER-2/neu oncogene expression and DNA ploidy in normal human kidney and renal cell carcinoma.

Using flow cytometry (FCM), we have investigated both the DNA content (stained with propidium iodide) and HER-2/neu oncogene expression (revealed by means of an anti-HER-2/neu monoclonal antibody) in neoplastic and non-neoplastic kidney samples from 20 patients with renal cell carcinoma. All the non-neoplastic samples and 15/20 (75%) renal cell cancers showed diploid modal DNA content while the remaining 5 neoplastic sample (25%) showed both diploid and hyperdiploid cell populations. In normal kidney the level of HER-2/neu oncoprotein was low (median fluorescence values in arbitrary units = 7.5 AU, range: 4-10 AU). In diploid renal cancers the level of HER-2/neu was slightly increased (median fluorescence values = 20 AU, range: 9.5-30 AU) (p < .005). The relationship of HER-2/neu expression to the cell cycle in these tumor samples is not clear since most of the cells express the antigen in all phases of the cell cycle. On the other hand, there is an association between HER-2/neu expression and abnormal DNA content suggesting that aneuploid pattern may be biologically related to overexpression of the HER-2/neu gene.