• Korean Journal of Medicinal Crop Science
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• v.5 no.3
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• pp.196-201
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• 1997

To characterize active cells during microspore culture of Angelica gigas, flow cytometric and epifluorescent techniques were applied. The knowledge obtained from these types of studies will give us insight into early stage in plant development and may lead to the application of microspore-derived from haploid plants for breeding in recalcitrant species. Viability of cultured microspore differed depending on the developmental stages. Frequencies of active cells from tetrad, uni-nucleate, bi-nucleate and matured pollen were 12.8, 49.3, 42.3 and 31.7%, respectively. Alive microspores have luminescent the green fluorescence stained with FDA and blue fluorescence stained with DAPI.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.5055-5061
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• 2014

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.17
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• pp.7201-7205
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• 2014

Chemokine and chemokine receptor expression by tumor cells contributes to tumor growth and angiogenesis and thus these factors may be considered as tumor markers. Here we aimed to characterize cells directly extracted from glioma, meningioma, and secondary brain tumors as well as non-tumoral cells in vitro. Cells were isolated from brain tissues using 0.2% collagenase and characterized by flow cytometry. Expression of SDF-1, CXCR4, CXCR7, RANTES, CCR5, MCP-1 and IP-10 was defined using flow cytometry and qRT-PCR methods. Brain tissue isolated cells were observed as spindle-shaped cell populations. No significant differences were observed for expression of SDF-1, CXCR4, CXCR7, RANTES, CCR5, and IP-10 transcripts. However, the expression of CXCR4 was approximately 13-fold and 110-fold higher than its counterpart, CXCR7, in meningioma and glioma cells, respectively. CXCR7 was not detectable in secondary tumors but CXCR4 was expressed. In non tumoral cells, CXCR7 had 1.3-fold higher mRNA expression than CXCR4. Flow cytometry analyses of RANTES, MCP-1, IP-10, CCR5 and CXCR4 expression showed no significant difference between low and high grade gliomas. Differential expression of CXCR4 and CXCR7 in brain tumors derived cells compared to non-tumoral samples may have crucial impacts on therapeutic interventions targeting the SDF-1/CXCR4/CXCR7 axis.

• Environmental Engineering Research
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• v.17 no.1
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• pp.35-39
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• 2012

In this study, multiparametric flow cytometry (FCM) was installed to enumerate the diagnosis of Pseudomonas aeruginosa ATCC 10145 and Escherichia coli K12 (IFO 3301). The nucleic acids (DNA/RNA) were double stained by a LIVE/DEAD bacLight viability kit, involving green SYTO 9 and red propidium iodide (PI), based on the permeability of two chemicals according to the integrity of plasma membrane. As the results showed, the gate for dead bacteria was defined as the range of $0.2{\times}10^0$ to $6.0{\times}10^1$ photo multiplier tube (PMT) 2 fluorescence (X-axis) and $2.0{\times}10^0$ to $2.0{\times}10^2$ PMT 4 fluorescence (Y-axis), and the gate for live bacteria was defined as the range of $6.0{\times}10^0$ to $6.0{\times}10^2$ PMT 2 fluorescence (X-axis) and $2.0{\times}10^0$ to $4.0{\times}10^2$ PMT 4 fluorescence (Y-axis). In the comparison of the number of the tested bacteria detected by FCM (viability assessment) and plate culture (cultivability assessment), the number of bacteria detected by FCM well represented the number of bacteria that was detected by the colony forming unit (CFU) counting method when bacteria were exposed to isopropyl alcohol and silver/copper cations. Consequently, it is concluded that the application of FCM to monitor the functional effect of disinfectants on the physiological status of target bacteria can offer more rapid and reliable data than the plate culture colony counting method.

• Proceedings of the Korean Society of Crop Science Conference
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• 1999.05a
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• pp.162-163
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• 1999

The goal of this research was (1) to describe the conditions and parameters required for the cell cycle synchronization and the accumulation of large number of metaphase cells in maize and other cereal root tips, (2) to isolate intact metaphase chromosomes from root tips suitable for characterization by flow cytometry, and (3) to construct chromosome-specific libraries from maize. Plant metaphase chromosomes have been successfully synchronized and isolated from many cereal root-tips. DNA synthesis inhibitor (hydroxyurea) was used to synchronize cell cycle, follwed by treatement with trifluralin to accumulate metaphase chromosomes. Maize flow karyotypes show substantial variation among inbred lines. thish variation should be sueful in isolating individual chromosome types. In addition, flow cytometry is a useful method to measure DNA content of individual chromosomes in a genotyps, and to detect chromosomal variations. Individual chromosome peaks have been sorted from the maize hybrid B73/Mol7. Libraries were generated form the DOP-PCR amplification product from each peak. To date, we have analyzed clones from a library constructed from the maize chromosome 1 peak. Hybridization of labeled genomic DNA to clone inserts indicated that $24\%,\;18\%,\;and\;58\%$ of the clones were highly repetitive, medium repetitive, and low copy, respectively. Fifty percent of putative low cpoy clones showed single bands on inbred screening, blots, and the remaining $50\%$ were low copy repeats. Single copy clones showing polymorphism will be mapped using recombinant inbred mapping populations. Repetitive clones are being characterized by Southern blot analysis, and will be screened by in situ hybridization for their potential utility as chromosome specific markers.

• Restorative Dentistry and Endodontics
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• v.22 no.1
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• pp.374-387
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• 1997

The purpose of this study was to examine the distribution of lymphocyte populations in normal, reversibly inflamed and irreversibly inflamed human dental pulp tissues using flow cytometry. Flow cytometry, with specific antibody and fluorochrome reagent allows us to know cellular properties of hematolymphoid cells by measuring fluorescence of stained cells. Before extirpation of pulps in routine endodontic treatment, the clinical diagnosis were performed by symptom. The extirpated pulp tissues were divided into normal pulp group (N=5), reversible pulpit is group(N=10) and irreversible pulpitis group(N=7). The specimen was placed into RPMI 1640 medium, minced into small pieces, and then digested in medium with collagenase. The cell suspension was resuspended in PBS for monoclonal antibody staining of T lymhocytes(CD3+), B lymphocytes (CD19+), T helper cell (CD4+) and T supressor cell (CD8+). The percentages of cells were counted by FACStar(BD) flow cytometer. Following results were obtained; 1. In the most normal and inflamed pulps, the percentages of T lymphocyte, B lymphocytes, T helper cell and T suppressor/cytotoxic cell were less than 1 % in total counted pulpal cells. 2. The higher percentages of T, B, T helper and T suppressor cells were observed in irreversible pulpitis group as compared with the normal pulp and reversible pulpitis group but the differences between groups were not statistically significant (p>0.05). 3. The percentages of T helper cells (CD4 + cells) were greater than that of T suppressor/cytotoxic cells (CD8 + cells) in the inflamed pulps.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.15 no.8
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• pp.5412-5418
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• 2014

The red seabream iridovirus (RSIV), which belongs to the iridoviridae, causes infectious fish diseases in many Asian countries, leading to considerable economic losses to the aquaculture industry. Using the yeast surface display (YSD) technique, a new experimental system was recently developed for the detection and identification of a variety of marine viruses. In this study, a coat protein gene of RSIV was synthesized based on the nucleotide sequence database and subcloned into the yeast expression vector, pCTCON2. The expression of viral coat proteins in the yeast strain, EBY100, was detected by flow cytometry and Western blot analysis. Finally, they were isolated from the yeast surface through a treatment with ${\beta}$-mercaptoethanol. The data suggests that the YSD system can be a useful method for acquiring coating proteins of marine viruses.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.253-257
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• 2005

This study was carried out to evaluate the effects of washing medium, breed and washing temperature of fresh and frozen-thawed boar sperm on mitochondrial activity and membrane integrity by flow cytometry. More than $80\%$ of fresh sperm washed with mTLP-PVA medium at $20^{\circ}C$ exhibited an intact membrane and a functional mitochondrion. With frozen-thawed samples, a large number of sperm showed both damaged membrane $(36.4\~46.9\%)$ and nonfunctional mitochondrion $(55.1\~71.1\%)$ in the mTLP-PVA and BTS washing media at $20^{\circ}C$. There were no breed effects of fresh and frozen-thawed sperm on mitochondrial activity and membrane integrity. The percentages of damaged membrane of fresh and frozen sperm, respectively, were higher at $4^{\circ}C$ washing temperature than at $20^{\circ}C$ washing temperature in the mTLP-PVA medium. We found that washing medium and washing temperature of fresh and frozen-thawed boar sperm were important for the analyses of mitochondrial activity and membrane integrity by flow cytometry.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.37-43
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• 2012

This study evaluated a method of sorting X and Y chromosomes based on size using the forward angle light scatter related refractive index (FSC) of a flow cytometer. Hanwoo bulls sperm were separated to X and Y chromosomes by the parameters of FSC or Hoechst 33342 intensity. As a result, using monitor program linked flow cytometry during sorting processing, the purities were $97{\pm}0.57$ or $96{\pm}0.67%$ for the X-fraction and $96{\pm}0.33$ or $97{\pm}1.33%$ for the Y-fraction in the two sperm sorting methods. There were no differences in the X and Y ratios (X and Y %) between the sperm sorting methods based on FSC or DNA content. The proportions of female and male embryos used for in vitro fertilization and development were $66.03{\pm}3.31$ or $69.37{\pm}1.41%$, and $70.56{\pm}2.42$ or $56.11{\pm}3.09%$ when sperm were processed using the sex sorting method by FSC or Hoechst 33342. In conclusion, further study is needed to determine the optimum procedure and improve the nozzle to enhancing sorting accuracy or efficiency. Also, the findings of this study do not negate the possibility that the difference method of sperm sorting cannot use a UV laser beam.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.22 no.2
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• pp.201-210
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• 1996

Predictive tests for the identification of contact sensitizing chemicals have been developed. We measured the sensitization potential with three predictive tests, the in vitro and the in vivo Local Lymph Node Assay(LLNA), ELISA to detect interferon-gamma(IFN-${\gamma}$) from supernatant and flow cytometry to detect change of cell surface proteins, using draining lymph nodes of mice. BALB/c mice were exposed to various chemicals or vehicles on the ears daily for 3 consecutive days in all experiments. With some exceptions of propyl paraben, neomycin sulfate, the in vivo LLNA was able to detect the sensitizing capacity of test chemicals and was more sensitive than the in vitro LLNA for chemicals used in the present study. In another experiment, contact sensitivity was assessed by the ELISA to detect IFN-Υ from the supernatants of the cultured LNCs after sensitization with chemicals. There was a good correlation between the LLNA and the IFN-Υ production for test chemicals. We also examined the change of cell surface proteins on LNCs after sensitization by flow cytometry for some cell adhesion molecules(ICAM-1, E-cadherine, B7 molecule), T cell markers(CD3, CD4, CD8, T$\alpha$$\beta$,T${\gamma}$$\delta$) and B cell markers(LR1, CD45R, I-Ad). The number of ICAM-1 positive cells and B cells in LNCs were increased after sensitization with DNCB, TNCB, isoeugenol and 25%, 50% cinnamic aldehyde compared with that of vehicle as a control. In conclusion, the in vivo LLNA could provide more sensitive screening test for moderate to strong sensitizers and some weak sensitizers including cosmetic raw materials than the in vitro LLNA. The production of IFN-Υ by allergen-activated LNCs might be a values indicators without radioisotopes for the identification of contact allergens. Detection of allergens by testing the increase of ICAM-1 positive cells and B cells in LNCs by flow cytometry might be used as a test method to detect allergens.