We developed large-scale preparation of phycoerythrin from Porphyra haitanensis, a main economic red algae in China. Firstly, P. haitanensis thallus was broken by using "swelling and smash" method. Then times of grads ammonium sulfate precipitation applied to the crude extraction were compared. Desalted solution was further purified with one-step chromatography using hydroxyapatite and properties on spectrum and molecular weight were identified finally. The results indicated that after four times of ammonium sulfate precipitation (15%, 50%, 10% and 40%), the absorption spectrum purity of P. haitanensis achieved 0.9 (A564/A280), and 507.82 mg phycoerythrin (A564/A280 > 3.2) was obtained from 7 kg fresh algae after further hydroxyapatite chromatography. This research provides a potential way for preparation of phycoerythrin in large sclae.
Ammonium Sulfate ; chemistry ; Chromatography ; methods ; Phycoerythrin ; isolation & purification ; Porphyra ; chemistry

Flow cytometry, a useful tool for measuring DNA content and cell differentiation as expressed by cell surface markers, is utilized to measure multiple antigens, especially surface antigen, intracellular oncoprotein, and DNA content, simultaneously. For this simultaneous detection, several methods off ixation and permeabilization have been used with limited values. In this study, 20 ng/ml of lysolecithin in 1% paraformaldehyde solution was utilized for fixation and permeabilization of cultured promyelocytic leukemic cells(HL 60). The cells were first stained with phycoerythrin (PE)-conjugated monoclonal antibody to the cell surface My 7 antigen and then were fixed and permeabilized with 20 ng/ml of lysolecithin in 1% partormaldehyde solution. After incubation, the fixed and permeabilized cells were stained with monoclonal antibody to intracellular c-myc antigen, which were followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The c-myc stained cells were finally stained for DNA content with 7-amino-actinomycin D(7-AAD). This procedure permits excellent staining for intracellular oncoproteins and preservation of surface antigens with relatively low cofficients of variation (CV) for the G0G1 peak of the DNA histograms and suggests that the sequential staining procedure of surface antigen, intracellular antigen, and DNA content will be extended for the study of correlations with cellular differentiation, expression of oncoproteins, and cell cycle analysis in the cells which are obtained from human malignant diseases using a 488 nm single laser flow cytometry.
Antigens, Surface* ; Cell Cycle ; Cell Differentiation ; DNA* ; Flow Cytometry* ; Fluorescein ; Humans ; Oncogene Proteins ; Phycoerythrin

OBJECTIVE: This study was designed to estimate the chemosensitivity by a quantitative evaluation of the apoptotic cell fractions using flow cytometry. METHODS: The OVCAR-3 cells were exposed to 20 nM or 30 nM taxol for 0 (control), 24 and 48 hours, then removed the taxol contained media, and cultured further with fresh media without taxol. (1) Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) were added to one test tube to detect the apoptotic cell fractions and at the same time, PI was added to the other tube to stain the DNA. (2) Annexin V-FITC and cytokeratin (clone CAM5.2 and MNF116) were added to the test tube. They were fixed and permeabilized with 1% paraformaldehyde solution and 100% methanol. They were then incubated with phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (GAM IgG1-PE or GAM IgG2a-PE) and sequentially stained with PI for DNA. All the stained cells were analyzed by a FACScan flow cytometer. RESULTS: (1) After treatment of 20 nM or 30 nM of taxol, G2M arrest was observed in both of treatment groups, which increased with time. (2) The G0G1 sub-fraction indicative of apoptosis increased with increase of culturing time from 24 hrs to 48 hrs. (3) The early apoptotic cell fraction with positive annexin V-FITC and negative PI increased with increase of culturing time. (4) In cells stained sequentailly with annexin V-FITC, cytokeratin (CAM5.2 and MNF116), and PI after 30 nM taxol treatment, the early apoptotic cell fractions increased with increase of culturing time. However, their extent was somewhat lower than those observed by positive annexin V-FITC and negative PI in cells treated with 20 nM of taxol. CONCLUSION: The results of sequential stainings with annexin V-FITC, cytokeratin, and PI were consistent with the those of annexin V-FITC and PI with parallel DNA staining. Our results suggested that the level of apoptosis detected by flow cytometry could be a marker of chemosensitivity which could select the sensitive anti-cancer agents before administration to gynecologic cancer patients.
Annexin A5* ; Apoptosis* ; Cell Line* ; DNA ; Evaluation Studies as Topic ; Flow Cytometry ; Fluorescein ; Goats ; Humans ; Immunoglobulin G ; Keratins* ; Methanol ; Ovarian Neoplasms* ; Paclitaxel ; Phycoerythrin ; Propidium*

OBJECTIVE: Taxol (paclitaxel)-induced apoptosis was studied to understand their biological mechanism correlated with the expression of p53 in the SKOV-3 and OVCAR-3 ovarian cancer cell lines. MATERIALS AND METHODS: The SKOV-3 and OVCAR-3 cell lines were cultured in RPMI 1640 medium without taxol (control group) and with taxol for 24 h and 48 h (experimental group). After harvest, the cells were stained with annexin V-FITC (fluorescein isothiocyanate) and anti-cytokeratin antibodies (clone CAM5.2 and clone MNF116). They were washed and stained with p53 antibody. After then the secondary antibodies, i.e., FITC- or phycoerythrin (PE)-conjugated goat anti-mouse (GAM) immunoglobulin G (GAM IgG-FITC or GAM IgG-PE) were added in the cells and they were incubated in the dark. DNA of these cells were stained sequentially with propidium iodide (PI). Standard FACScan equipped with a 488 nm single laser was used for the analysis of these cells. RESULTS: Both of SKOV-3 and OVCAR-3 cell lines were arrested in the G2M phase after treatment of taxol, suggesting that these cells would eventually enter into the stage of cell death. Fractions of negative cytokeratin and positive annexin V and amount of sub-G0G1 fraction indicative of apototic fractions were lower in the SKOV-3 cell line compared with that in OVCAR-3 cell line, probably as a result of lower sensitivity of SKOV-3 cell line to the taxol. p53 expression were not detected in SKOV-3 cell line. On the basis of observed findings in SKOV-3 cell line and findings of high expressions of p53 regardless of taxol treatment, no increases in their expressions according to culturing time, and gradual increases in sub-G0G1 fractions and in fractions of negative cytokeratin and positive annexin V indicative of apoptosis in OVCAR-3 cell line, we concluded that the expression of p53 would not be associated with cell cycle changes and the arrest in the G2M pahse but associated with the appearance of apotosis. CONCLUSION: Our results suggest that flow cytometric detection of the apoptotic fractions would be an effective, fast, and accurate method for the chemosensitivity test in tumor cells before the administration of anti-cancer drugs in gynecologic cancer patients.
Annexin A5 ; Antibodies ; Apoptosis* ; Cell Cycle ; Cell Death ; Cell Line* ; Clone Cells ; DNA ; Flow Cytometry ; Goats ; Humans ; Immunoglobulin G ; Keratins ; Ovarian Neoplasms* ; Paclitaxel ; Phycoerythrin ; Propidium

BACKGROUND AND OBJECTIVES: The thioredoxin (TRx) system is a ubiquitous thiol oxidoreductase pathway that regulates cellular reduction/oxidation status. Although endothelial cell (EC) hypoxic damage is one of the important pathophysiologic mechanisms of ischemic heart disease, its relationship to the temporal expression pattern of the TRx system has not yet been elucidated well. The work presented here was performed to define the expression pattern of the TRx system and its correlation with cellular apoptosis in EC lines in hypoxic stress. These results should provide basic clues for applying aspects of the TRx system as a therapeutic molecule in cardiovascular diseases. SUBJECTS AND METHODS: Hypoxia was induced with 1% O2, generated in a BBL GasPak Pouch (Becton Dickinson, Franklin Lakes, NJ, USA) in human endothelial progenitor cells (hEPC) and human umbilical vein endothelial cells (HUVEC). Apoptosis of these cells was confirmed by Annexin-V: Phycoerythrin flow cytometry. Expression patterns of TRx; TRx reductase; TRx interacting protein; and survival signals, such as Bcl-2 and Bax, in ECs under hypoxia were checked. RESULTS: Apoptosis was evident after hypoxia in the two cell types. Higher TRx expression was observed at 12 hours after hypoxia in hEPCs and 12, 36, 72 hours of hypoxia in HUVECs. The expression patterns of the TRx system components showed correlation with EC apoptosis and cell survival markers. CONCLUSION: Hypoxia induced significant apoptosis and its related active changes of the TRx system were evident in human EC lines. If the cellular impact of TRx expression pattern in various cardiovascular tissues under hypoxia or oxidative stress was studied meticulously, the TRx system could be applied as a new therapeutic target in cardiovascular diseases, such as ischemic heart disease or atherosclerosis.
Anoxia ; Apoptosis ; Atherosclerosis ; Cardiovascular Diseases ; Cell Hypoxia ; Cell Survival ; Endothelial Cells ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; Humans ; Lakes ; Myocardial Ischemia ; Oxidative Stress ; Phycoerythrin ; Stem Cells ; Thioredoxins

We studied the effect of photodynamic therapy with phycobiliproteins on human liver cancer cells in vitro. With 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), we used two phycobiliproteins, R-phycoerythrin (R-PE) and C-phycocyanin (C-PC) prepared from Porphyra yezoensis, to determine the killing rates and apoptosis rates of human liver cancer cells (SMMC-7721) mediated by laser. When the concentration of R-PE was 120 mg/L, the survival rate of human liver cancer cells was 27% after treated by Argon laser with 100 J/cm2 doses, while the survival rate in the control group (without adding R-PE) was 65%. When the C-PC concentration was 120 mg/L, the survival cell rate was 47% after treated by He-Ne laser with 35 J/cm2 dose, while the survival rate in the control group (without adding C-PC) was 70%. After handled only with these two kinds of phycobiliproteins for 72 h, the growth of cancer cells presented significant inhibition. The maximal inhibition rates reached up to 31% with R-PE (120 mg/L concentration) and 27% with C-PC (250 mg/L concentration) respectively. After irradiated by laser for 8 h, the maximal cell apoptosis rates were 31.54% with R-PE and 32.54% with C-PC, respectively. It indicated that R-PE and C-PC extracted from Porphyra yezoensis could develop to new photosensitizers for cancer photodynamic therapy.
Apoptosis ; drug effects ; radiation effects ; Cell Line, Tumor ; Humans ; Lasers ; Liver Neoplasms ; pathology ; Photochemical Processes ; Photochemotherapy ; methods ; Phycobiliproteins ; isolation & purification ; pharmacology ; Phycoerythrin ; isolation & purification ; pharmacology ; Porphyra ; chemistry

BACKGROUND: Chronic neuropathic pain is often associated with altered immune function and the modulated immune cell response play a role in neuropathic pain by experimental nerve injury. In order to assess the possible changes in lymphocytes function following peripheral mononeuropathy, this study examined the lymphocyte subpopulation of the spleen using the monoclonal antibodies against the membrane surface markers in neuropathic BALB/c mice by a partial transection of sciatic nerve (PST). METHODS: After confirming tactile allodynia by paw withdrawal threshold, the splenic lymphocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD45R/B220 (B cell) and CD4 (helper/inducer T cell) or with phycoerythrin (PE)-conjugated anti-mouse CD90.2 (total T cell) and CD8 (suppressor/cytotoxic T cell). The proportions of subsets were analyzed using a FACScan laser flow cytometry system on postoperative day 5 and day 18 respectively. RESULTS: PST induced a mechanical allodynia as verified by the von Frey test at both 5 and 8 days postoperatively compared to pre-surgery (P < 0.05). Lymphocyte subpopulation was affected by PST. The proportion of CD4+ subset was significantly larger in the PST group than in the sham operated group on day 5, while the proportion of CD8+ subset was larger on day 18. In the PST group, there were significantchanges in the proportion of CD4+ on day 5 and in the proportion of CD8+ on day 18 (P < 0.05) compared to pre-surgery. There were no significant fluctuations in the proportion of total splenic T cell and B cell subsets of PST group compared to sham operated group. CONCLUSIONS: These results suggest that development of mononeuropathy is responsible for the proportional changes in splenic lymphocyte subsets in mice.
Animals ; Antibodies, Monoclonal ; B-Lymphocyte Subsets ; Flow Cytometry ; Fluorescein ; Hyperalgesia ; Lymphocyte Subsets* ; Lymphocytes* ; Membranes ; Mice* ; Mononeuropathies ; Neuralgia ; Peripheral Nerve Injuries* ; Peripheral Nerves* ; Phycoerythrin ; Sciatic Nerve ; Spleen*

PURPOSE: Multiparametric flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic and ploidy heterogeneity of tumor cell populations. But there are major problems such as inaccurate results by the contribution of non-neoplastic cell contamination and the substantial spectral overlap of PI (propidium iodide) into PE (phycoery- thrin) fluorescent emissions on a standard flow cytometer. Recent studies suggested that the emission spectral overlap from PI into PE could be sufficiently compensated electrically and the cytokeratin, a marker for epithelial tumor cells, are successfully used in conjunction with DNA specific dye so as to obtain DNA profiles selectively for cytokeratin-positive tumor cells. The aim of this study was to investigate the feasibility that multiparametric analysis in heterogeneous cell populations of cell lines like solid tumors, which were stained triply with PE, fluorescein isothiocyanate FITC, and PI, can be done without any influences by the contaminated normal diploid cell populations and without spectral overlap between fluorochromes on a standard flow cytometer. MATERIALS AND METHODS: MCF-7 cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes were fixed with 1% paraformal- dehyde and permeabilized with 100% methanol. Cytokeratin was labeled with PE and some proliferat!on-associated markers were labeled with FITC, which were followed by DNA staining by PI. These triply stained cells were measured on a standard FACScan flow cytometer equipped with 488 nm single laser and those acquired data were analyzed with WinList 3.0 and ModFit LT software programs on personal computor. RESULTS: Coefficient of variation (CV) of GoG1> peak of MCF-7 cells alone was 4.3. GoG1, S, and G2M phase fractions were 44.9%, 45.9%, and 9.2% respectively. FITC, PE and PI fluorochromes could be detected without any interference between them. CVs of GoG1 peak of PBL and MCF-7 cells in those heterogeneous population were 2.3 and 4.2 respectively. The DNA index of MCF-7 cells was 1.7. MCF-7 cells expressed the cyto- keratin, PCNA, p53, c-erbB/2 and c-myc antigen and in contrast, PBL did not express cytokeratin. The cell cycle phase fractions and oncoprotein expressions could be detected separately in diploid PBL and aneuploid MCF-7 cells in the mixed cell population without any influences by each other. CONCLUSION: These results suggested that the cellular antigen expressions of the malignant cells can be analyzed selectively without influences of fluorescent signals from nonneo- plastic cells. The neoplastic tumor subpopulations are clearly identified on the basis of both ploidy status and antigen expressions. The positive cytokeratin expressions indicate that they were derived from the epithelium, providing objective evidence of the tissue of origin and more precise analysis of DNA contents, ploidy, and oncogene expressions selectively with possible correlation between them. Thus, this method offers new possibilities for multiparameter flow cytometric analysis in the heterogeneous solid tumor cell populations.
Aneuploidy ; Breast Neoplasms* ; Breast* ; Cell Cycle ; Cell Line* ; Diploidy ; DNA ; Epithelium ; Flow Cytometry* ; Fluorescein* ; Fluorescein-5-isothiocyanate ; Fluorescent Dyes ; Humans ; Keratins ; Lymphocytes ; MCF-7 Cells ; Methanol ; Oncogenes ; Phycoerythrin* ; Plastics ; Ploidies ; Population Characteristics ; Proliferating Cell Nuclear Antigen ; Propidium*

BACKGROUND: HLA-B27 is associated with an increased incidence of specific spondyloarthropathies(SpA), most notably ankylosing spondylitis(AS). I evaluated the cases referred for HLA-B27 antigen using flowcytometry (FCM) to find the clinical characteristics of the patients and the diagnostic utilities of median fluorescence intensity (MFI) in HLA-B27 program. METHODS: I evaluated 443 subjects of HLA-B27 cases using FACScan flowcytometry, consisted with software for automated calibration and analysis, calibration beads, and the anti-HLA- B27 fluorescein isothiocyanate (FITC)/anti-CD3 phycoerythrin (PE) monoclonal antibodies (all from Becton Dickinson, San Jose, CA). RESULTS: Of the total 443 cases, the positive rate in male cases was 44% (132/300) and it was higher than that of female cases (22.4%, 32/143). The gating procedure was failed in one sample of 443 (0.23%). The positive rates in each diagnostic criteria were as follows; AS 61.6%, gout 20.0%, herniated intervertebral disc 20%, lower back pain 25.6%, polyarthritis 16.0%, psoriatic arthritis 20.0%, rheumatoid arthritis 28.3%, reactive arthritis 26.9%, SpA, undifferentiated 31.8% and uveitis/iritis 23.8%. In AS group, 89 cases (95.7%) showed MFI values higher than 150. CONCLUSION: About 62% of AS group showed HLA-B27 positivity using FCM and the positive rates of other diseases group in SpA categories were around 20-30%. If we considered MFI value 150 as differential value, about 95% of HLA-B27 positive AS cases might not need further confirmatory study to differentiate HLA-B7.
Antibodies, Monoclonal ; Arthritis ; Arthritis, Psoriatic ; Arthritis, Reactive ; Arthritis, Rheumatoid ; Calibration ; Female ; Fluorescein ; Fluorescence ; Gout ; HLA-B27 Antigen* ; HLA-B7 Antigen ; Humans ; Incidence ; Intervertebral Disc ; Low Back Pain ; Male ; Phycoerythrin ; Spondylarthropathies ; Spondylitis, Ankylosing

BACKGROUND: CD34+ cells are capable of engraftment and hematopoietic reconstitution. However, expression patterns of other surface antigens such as CD13, CD33, CD38 and HLA-DR on CD34+ cells in mobilized peripheral blood have remained unclear. This study analyzed the expansion kinetics of the CD34+ cells and subsets in the peripheral blood of healthy donors treated with recombinant human Granulocyte Colony-Stimulating Factor (rhG-CSF), the relative composition of CD34+ subsets in mobilized peripheral blood and apheresis product, the yield of apheresis product. METHODS: The 6 peripheral blood stem cell donors received a daily dose of 500 microgram (8.1~10 microgram/kg) of rhG-CSF subcutaneously for 6 or 7 days. The hematologic parameters and the number of CD34+ cells and subsets in peripheral blood were recorded at baseline and daily for a total 6 to 7 days. With monoclonal antibodies which were designed for two color direct immnunofluorescence (IF) analysis with combination of fluorescence isothiocyanate (FITC) and phycoerythrin (PE) conjugated, CD34CD38, CD34HLA-DR, CD34CD13, and CD34CD33 surface antigens were analyzed by flow cytometry. RESULTS: The number of CD34+ cells mobilized to peripheral blood peaked at day 4 or 5 with rhG-CSF treatment. Circulating CD34+ cell expanded by 11.5-fold from 4.5 +/- 1.8x106/L before rhG-CSF treatment to maximal increment 51.9 +/- 13.4x106/L after mobilization. Subsets of CD34+CD38-, CD34+HLA-DR-, CD34+ CD13-, and CD34+CD33- (in % ofthe CD34+ cell) were all decreased and subsets of CD34+CD38+, CD34+HLA-DR+, CD34+CD13+, and CD34+CD33+ were all increased after mobilization, so that the numbers of early committed and myeloid committed progenitors were higher than the those of multipotent stem cells in mobilized peripheral blood and leukapheresed products. Each apheresis product yielded a mean of 451.6x106 (7.7x106/kg of RBW) CD34+ cell and 172.3x105 (2.9x105/kg of RBW) CFU-GM. CONCLUSION: Sufficient amount of CD34+ cells capable of engraftment and hematopoietic reconstitution can be mobilized by administration of rhG-CSF to healthy adult donor. Subsets of mobilized CD34+ cells were mainly composed of early committed and myeloid committed progenitors, although absolute numbers of all progenitor cells including multipotent stem cells were significantly increased.
Adult* ; Antibodies, Monoclonal ; Antigens, Surface ; Blood Component Removal ; Flow Cytometry ; Fluorescence ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells ; HLA-DR Antigens ; Humans* ; Kinetics ; Multipotent Stem Cells ; Phycoerythrin ; Stem Cells ; Tissue Donors*