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PubMed1998

Characterization of dnaC2 and dnaC28 mutants by flow cytometry.

Escherichia coli strains containing thermosensitive dnaC alleles were studied by flow cytometry. Strains containing either the dnaC2 or dnaC28 allele were shifted between different temperatures, and DNA content distributions were gathered. Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters. These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration. Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants. Efficient use of dnaC mutants for cell cycle studies is discussed.

Bacterial Proteins↗

PubMed1988

Retrospective study of prognostic importance of DNA flow cytometry of urinary bladder carcinoma.

Cellular DNA content was determined by flow cytometry on routinely processed paraffin sections of 61 primary and untreated transitional cell carcinomas of the urinary bladder, and correlated with tumour grade and stage and clinical follow up. All 16 (25%) grade 1 carcinomas were diploid and all 11 (20%) grade 3 tumours were aneuploid. The 34 (55%) grade 2 carcinomas comprised 13 (40%) diploid and 21 (60%) aneuploid cases. Among the 37 superficial carcinomas (stage Ta and T1), 25 (65%) were diploid; 20 (85%) of the 24 advanced tumours (stage T2 to T4) had aneuploid tracings. Ploidy was a significant prognostic indicator (p: 0.006) of five year survival. The initial presence of aneuploidy in superficial bladder carcinoma (stage Ta and T1) is a strong argument for more aggressive treatment than is customary.

Aged↗

PubMed1984

The use of multiparameter flow cytometry to assess tumor cell heterogeneity and grade prostate cancer.

Multiparametric flow cytometry has permitted the quantitative assessment of tumor cell heterogeneity within a single tumor. This technique was applied to four tumor lines with biological activity similar to the variety observed in human prostate cancer. These tumors were derived from the Dunning R3327 transplantable rat prostatic adenocarcinoma and have permitted the development of an index to assess, quantitate, and discriminate variation in the aggressive nature of these tumors. The index is based upon measurements of the mean and the variance for perpendicular light scatter and forward light scatter. Perpendicular light scatter has been shown to correlate with nuclear shape, volume, and roundness as measured by digitized image analysis. Forward light scatter increases in quantity and range as the tumors become more undifferentiated. This technique is rapid, reproducible, and applicable to the quantitative measurement of cytometric parameters on large numbers of individual tumor cells.

Adenocarcinoma↗

PubMed1990

Evaluation of DNA flow cytometry as a screening test for bladder cancer.

We evaluated DNA flow cytometry of urine histogram profiles as a screening procedure for bladder cancer. To establish the false-positive rate, we first studied a low-risk group of 90 patients with no history of bladder cancer: 24 with benign prostatic hyperplasia and 66 with upper tract stones, and found 4.4% unsatisfactory and 15.1% abnormal histograms. Urine samples were then obtained for 54 additional patients (most being evaluated for benign prostatic hyperplasia) whose selection was based on their smoking habits. These included 20 nonsmokers, 10 who had not smoked for at least 20 years (mean: 18 packs/year) and 24 current smokers (mean: 71 packs/year). Abnormal histograms were obtained for 14% of nonsmokers and for 36% of smokers, a group known to be at risk for developing bladder cancer. Although abnormal histograms are more frequent for smokers, the background false-positive rate remains unacceptably high for general screening purposes.

Carcinoma, Transitional Cell↗

PubMed1996

Differential effects of 3,5,3'-triiodothyronine on control and mdx myoblasts and fibroblasts: analysis by flow cytometry.

The possibility of differential effects of triiodothyronine (T3) treatment in vivo on myoblast and fibroblast cell proliferation was examined in control and mdx muscle cultures. Cell isolates were purified in a Percoll gradient, sorted by flow cytometry (light scatter), and characterized as myoblasts and fibroblasts using antiskeletal muscle myosin fluorescence. The two cell types were grown separately or remixed (1:1). Cultures were incubated with or without T3 (10(-9) M) for 19 h. Cells were either exposed to [3H]thymidine for 1 h and DNA prepared for scintillation counts or stained with propidium iodide for cell cycle analysis by flow cytometry. Overall [3H]thymidine uptake per cell was greater in mdx than control cells (mainly fibroblasts and mixed cells) and was decreased by T3 only in myoblast and mixed cultures. Cell cycle data showed that the effects of T3 originated primarily at the G0/G1 phase. There were more mdx than control myoblasts at G0/G1 without T3. After T3 treatment, more control fibroblasts than myoblasts were at G0/G1, but more mdx myoblasts than fibroblasts were at G0/G1. In the absence of T3, there were also fewer mdx than control myoblasts in S. After T3, only the proportion of mdx myoblasts in S phase was reduced. Results are consistent with distinct T3 effects on muscle regeneration in vivo and support the hypothesis that cycling and proliferation of mdx and control myoblasts are differentially modulated by T3. As control and mdx fibroblasts also showed distinct responses to T3, muscle regeneration likely occurs by a complex regulation of gene expression endogenous to specific cell types as well as interactions between cells of different lineage.

Animals↗

PubMed2001

A simple and highly efficient fixation method for Chrysochromulina polylepis (Prymnesiophytes) for analytical flow cytometry.

BACKGROUND: To study the fragile Prymnesiophyte species Chrysochromulina polylepis by flow cytometry (FC), we needed an effective fixation method. This method must guarantee a high yield of fixed cells to achieve acceptable measurement times by FC and to allow quick processing of many samples. Moreover, we wanted a method that allows for storage of fixed samples when FC analysis cannot be done immediately. METHODS: Different aldehydes and methanol were tested at different final concentrations. Gravity sedimentation and centrifugation were applied to achieve higher cell concentrations. Storage of fixed samples was tested under different conditions. RESULTS: 0.25% glutaraldehyde (GA) fixation yielded a recovery rate of about 90%. The signals obtained by FC analysis were excellent. It is possible to centrifuge GA-fixed cells and to store them for several weeks. CONCLUSIONS: GA is the fixative of choice for FC analysis of C. polylepis (and possibly other small delicate species) because it yielded highly significant recovery rates and high-quality FC signals. Cells can be centrifuged to increase the cell concentration, thereby achieving short measurement times with FC. The possibility of long-term storage of fixed cells presents an additional advantage if FC analysis cannot be done immediately.

Aldehydes↗

PubMed2003

Reevaluation of flow cytometry for investigating antibody binding to the surface of Plasmodium falciparum trophozoite-infected red blood cells.

BACKGROUND: The acquisition of antibodies directed toward variant surface antigens (VSAs) expressed on the surface of the trophozoite-infected red blood cell is an important determinant of natural immunity to Plasmodium falciparum malaria. In recent years, flow cytometry has been used increasingly to investigate these responses, but few systematic assessments of this method are available in the published literature. METHODS: We developed a highly standardized experimental protocol and used parasites of the A4 laboratory clone, a monoclonal antibody to the VSA expressed by this clone (monoclonal antibody BC6), and a single pool of hyperimmune plasma to explore the parameters responsible for variations in VSA antibody responses measured by flow cytometry. RESULTS: Despite strenuous efforts to standardize our flow cytometric assay, we found marked variability in our assay readout, even between repeat experiments using identical antibody and parasite combinations. We found no remediable cause for much of this variability. However, we identified three major factors that we considered important contributors: antibody concentration, nonspecific antibody binding to uninfected red blood cells, and parasite agglutination. CONCLUSIONS: A number of potential pitfalls should be considered when designing and interpreting studies using this technique. In particular, we suggest that comparisons between assays conducted on different occasions can be made only through reference to carefully selected standards. We anticipate that a better appreciation of the factors that lead to assay variation will assist the design of improved experimental protocols.

Animals↗

PubMed1990

[Application of flow cytometry to the study of hematologic disorders: analysis of oncogene products].

Flow cytometry (FCM) is a useful method for clinical research of oncogene products since it can analyze proteins quantitatively which are located at cell surfaces or inside of cells. Oncogene products are now under study by FCM not only as tumor markers but also as functioning proteins in carcinogenesis. The examples of oncogene products analyzed by FCM are ras, myc, p53, myb and fos; those of cell-proliferation-related proteins are Ki-67, PCNA and DNA polymerase alpha. In some diseases the relationship between these proteins and disease classification, stage, pathophysiology, or prognosis have been clarified. Using dual color FCM of H-ras p21 and DNA, we analyzed the expression of H-ras p21 in human multiple myeloma and leukemias and found that H-ras p21 levels in multiple myeloma strongly correlated to the prognosis of patients (p = 0.03). When AML cells were stimulated by adding G-CSF, it was found that many cells proliferated but some were dying. The percentage of dying cells was small in one AML case whose myeloblasts showed increased expression of H-ras p21 by G-CSF stimulation. Together with other papers reviewed, it is conceivable that H-ras p21 expression is related to cell proliferation and inhibition of cell autolysis. Thus FCM is useful in the classification of the role of oncogene products in carcinogenesis in clinical cases.

Biomarkers, Tumor↗

PubMed1992

Selective detection of human hepatitis B virus surface and core antigens in some peripheral blood mononuclear cell subsets by flow cytometry.

The presence of HBs and HBc antigens was investigated, by flow cytometry, on the surface of peripheral mononuclear cells (PBMC) from the following phenotype: CD3 (T lymphocytes), CD4 (T helper/inducer), CD8 (T cytotoxic/suppressor), CD19 (B lymphocytes) and CD56 (NK cells) among 8 patients suffering from chronic hepatitis B and 5 healthy HBV-negative subjects. This study demonstrated the presence of HBsAg and HBcAg on the lymphocyte surface for most of the patients. The mean percentage of labelled cells was 17% for HBsAg and 15% for HBcAg. Among the different lymphocyte subsets only B lymphocytes and the NK cells expressed HBsAg for 57% and 26% of cells, respectively. Similarly HBcAg was also detected among CD19 and CD56 cells only. PCR was used to search for the presence of HBV DNA and RNA in PBMC, using primers located in the S gene. HBV DNA was detected with variable intensity in the CD3, CD4, CD19 and CD56 subsets following their separation with a cell sorter. For HBV RNA the signal obtained after PCR and Southern blotting was higher for CD56 and CD19 cells than for CD3 cells and undetectable for CD4 cells. This study demonstrates that replication and transcription can occur in CD19 and CD56 cells. Positive signals in CD3 cells may possibly be due to contamination of this subpopulation by NK cells.

Antigens, CD↗

PubMed1990

Deoxyribonucleic acid flow cytometry in predicting response to radical radiotherapy of bladder cancer.

Using flow cytometry tumors can be classified according to their deoxyribonucleic acid content as diploid or aneuploid with 1 or several cell lines and to the ploidy level of the aneuploid cell lines, as well as to the proportion of proliferating cells. We used this technique in 73 patients with invasive transitional cell carcinoma of the bladder who received radical radiation therapy with followup of surviving patients for at least 5 years. The effects of therapy were related to the deoxyribonucleic acid patterns before irradiation. All diploid tumors disappeared, while aneuploid tumors with 1 cell line disappeared in 55% and those with several cell lines in 30% of the patients. Aneuploid tumors with cell lines exceeding 4c appeared to be more radiosensitive than those in the triploid region. Thus, tumors unresponsive to therapy as well as those that recurred after irradiation were mostly triploid. Since most of the tumors had high proliferation rates the proportion of S-phase cells was of only limited value for further classification of the various tumors. In the choice between radical radiotherapy, in which bladder function is maintained, and cystectomy an analysis of tumor ploidy provides at least some basis on which radiation response can be predicted. In the choice of therapy we must also consider the fact that almost 20% of the patients who failed to respond to therapy revealed aneuploidy only in bladder washings after irradiation, indicating existence of concomitant carcinoma in situ.

Aged↗

PubMedDate not supplied

Anticancer drug action assessed by serial DNA flow cytometry during induction chemotherapy in oral squamous cell carcinoma: impacts on the development of individualized treatment strategies.

Biopsy specimens from 53 advanced oral squamous cell carcinomas were taken before and after intra-arterial induction chemotherapy and analyzed by DNA flow cytometry. The 5-year overall survival rate was 90% in patients with diploid carcinomas in contrast to only 18% of those with pretherapeutically aneuploid tumors. A clear decrease of aneuploid cell numbers during chemotherapy indicated that DNA flow cytometry substantially allows monitoring of cytotoxic effects in vivo. Even if the immediate response to chemotherapy was significantly better in carcinomas with a complete disappearance of aneuploid tumor cells during treatment than in cases with persisting aberrant cells, the overall survival rate was nearly identical in both groups.

Aneuploidy↗

PubMed1985

Progressive muscular dystrophy (Duchenne): biochemical studies by flow-cytometry.

The peanut lectin (PNL) receptor density of the cell membrane and several metabolic parameters of cultured fibroblasts of normal human individuals and of patients with muscular dystrophy were measured by simultaneous two and three parameter flow cytometry. The PNL-receptor density was significantly decreased on muscular dystrophy fibroblasts (between 20.7 and 33.6%) as compared to normal fibroblasts. The cell volume, the esterase activity, the intracellular pH, and the percentage of proliferating cells of both types of fibroblasts were not significantly altered. The mean cell volume of different fibroblast cultures varied between 2500 and 6000 micron 3. The concentration of the intracellular esterase activity of fibroblasts was low (0.169 relative units) as compared to lymphocytes and granulocytes of the peripheral blood (1.56 and 2.17 relative units). The fibroblasts had an acidic intracellular pH of 6.52 while lymphocytes and granulocytes had basic pH values of 7.30 and 7.17. Some of the fibroblasts were in the S + G2/M phase of the cell cycle (20%). The study shows that the measurement of biochemical parameters of vital and fixed single fibroblasts by flow-cytometry is of great interest for the recognition of differences between normal individuals and muscular dystrophy patients.

Cell Cycle↗

PubMed1998

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry.

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.

Animals↗

PubMed2004

Bacteriological examination of ballast water in Singapore Harbour by flow cytometry with FISH.

In this study the concentrations of total bacteria, enterobacteria, Vibrio spp., and E. coli have been compared for ballast water samples taken from ships in Singapore Harbour. The cell concentrations were enumerated using FISH and flow cytometry. The data were highly variable, reflecting the many influences upon ballast water as it is utilized in the shipping industry. The concentration of bacterial species was determined as a proportion of the total concentration of cells for the ballast water sampled. For the ballast water sampled these concentrations were 0.67-39.55% for eubacteria, 0-2.46% for enterobacteria, 0.18-35.82% for Vibrio spp., and 0-2.46% for E. coli. Using FISH and flow cytometry, an informative determination of the bacterial hazards of ship ballast water can be made.

Bacteria↗

PubMed2006

Evolutionary algorithms and flow cytometry to examine the parameters influencing transconjugant formation.

An evolutionary algorithm was used to determine the optimal combination of parameters for transconjugant formation. As a model system, a gfp tagged TOL plasmid pWW0 was chosen to examine transfer from Pseudomonas putida to Escherichia coli. A comparison of flow cytometry results with plating and microscopy showed that the majority of transconjugants were not culturable. The transconjugant ratio therefore was determined by flow cytometry. The evolutionary algorithm showed that the optimal conditions were obtained at 28 degrees C and at the highest nutrient concentrations. This work demonstrates that evolutionary algorithms can be used to find optimal parameter interactions in environmental microbiology.

Algorithms↗

PubMed1986

Comparative analysis of surface membrane immunoglobulin determination by flow cytometry and fluorescence microscopy.

The analysis of membrane surface immunoglobulin (SmIg) on B lymphocytes was carried out in 59 normal individuals and nine patients with B-cell non-Hodgkin's lymphomas by conventional immunofluorescence microscopy and flow cytometry. Five channel settings of a cytofluorograph were evaluated (100, 150, 200, 250, 300) and the mean and standard deviation of the percent positive cells were calculated and compared to the mean and standard deviation of the microscope reading. On the basis of the relative fluorescence reactivity, we were able to determine a fluorescence intensity at which the results of flow cytometry and fluorescence microscopy were comparable. In normal individuals, for cells expressing surface Ia, the channel giving similar results to that of fluorescence microscopy was 150; for kappa and lambda chains, channel 200; for Fab'PV, channel 200; and for IgM, channel 250. In patients with B-cell non-Hodgkin's lymphomas, for cells expressing surface Ia the channel giving similar results to that of fluorescence microscopy was 100; for kappa, channel 100; for lambda, channel 200; for Fab'FV, channel 150; and for IgM, channel 150. Flow cytometric analysis of SmIg appears to be superior to fluorescence microscopy in efficiency, and has the added advantages of being a rapid, sensitive, and objectively quantitative methodology.

B-Lymphocytes↗

PubMed2003

A flow cytometry technique to study nuclear factor-kappa B (NFkappaB) translocation during human B cell activation.

We aimed at examining NFkappaB translocation in B lymphocytes during in vitro activation through the specific receptor for antigen using a technique convenient in most laboratories such as flow cytometry. We present here an original, convenient, and reproducible technique to study B cell activation events through NFkappaB translocation by means of a novel, specific flow cytometry assay. Intranuclear translocation of NFkappaB p65 was induced after a 45min stimulation; the highest signal was detected for a 10 ng/ml stimulus compared to the unstimulated condition (P< 0.05). Purified CD19+ B cells--cultured in the presence of optimal concentrations of anti-micro fragment Abs (10ng/ml) for 45min at 37 degrees C--induced a mean 60% (range: 45-67%) MFI-- and thus, nuclear NFkappaB translocation-increase. We observed a one-pike profile of NFkappaB staining in PBMC B cells and a two-pike profile of NFkappaB staining in using tonsil B cells. B cells are susceptible to various dysregulations leading to minor to severe pathology (including immunoproliferative disorders). Studies of signal transduction carried out specifically in human B cells, using a novel technique gave considerable advantages: feasibility, sensitivity, reproducibility, ease.

Antigens, CD19↗

PubMed2003

Analysis of high-pressure-induced disruption of human erythrocytes by flow cytometry.

High-pressure-induced hemolysis is suppressed by pretreating human erythrocytes at 49 degrees C, or enhanced by pretreatment with trypsin. So, the response of these pretreated cells to a pressure of 200 MPa was examined using flow cytometry. In the case of intact erythrocytes, a major product was fragmented particles. From 49 degrees C-pretreated cells, vesicles were mainly released. Trypsin-pretreated cells mainly produced open ghosts. Additionally, intact erythrocytes, 49 degrees C-pretreated ones, and trypsin-pretreated ones also released at 200 MPa vesicles of diameter 464 +/- 9, 259 +/- 18, and 574 +/- 16 nm, respectively. These results suggest that mother cells, fragmented particles, vesicles, and open ghosts from 200 MPa-treated erythrocytes are easily monitored by flow cytometry and that the size of released vesicles may also be an important factor in high-pressure-induced hemolysis.

Air Pressure↗