BACKGROUND: Simple manual method using a Nageotte hemocytometer counting residual leukocytes [White blood cells(WBC)] in blood components is subjective, labor-intensive, time consuming and variable within and between laboratories. The aim of this study was to evaluate usefulness for flow cytometric method to determinate very low numbers of leukocytes in leukocyte-free blood components. METHODS: Epics XL-MCL (Beckman Coulter Co.) was used for determination of fluorescence-labelled cells. The DNA in leukocytes was stained using propidium iodide (PI) and leukocytes were automatically analysed by flow cytometer. RESULTS: Linearity determined over a range of 0.5-50 WBC/muL was a value of r=0.994. The detection limit of this method was 4.4 WBC/muL and accuracy was 86.6% with linearity of r=0.991 over the 5-50 WBC/muL. Reproducibility was CV of 9.1% for 25.8 WBC/muL and 14.7% for 7.1 WBC/muL, respectively. CONCLUSION: Flow cytometric techniques provide a reproducible and objective tool for counting residual WBC in leukocyte free blood components compared with the Nageotte hemocytometer.
DNA ; Leukocytes* ; Limit of Detection ; Propidium

OBJECTIVE: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. MATERIALS AND METHODS: A total 964 blastocysts (early~hatched) was exposed to PI (n=831) (group I: Animals ; Bisbenzimidazole* ; Blastocyst* ; Mice* ; Propidium*

To determine whether mitomycin-C[MMC]treatment induces apoptosis in cultured keratocytes. Cultured rabbit keratocytes were exposed to MMC[up to 0.4 milligram/milliliter]or phosphate-buffered saline[PBS]for 5 minutes. Light and transmission electron microscopic examination, DNA electrophoresis, and flow cytometric analysis with propidium iodide staining were performed 24 hours after MMC or PBS treatment. A characteristic findings consistent with apoptosis were observed under both light and electron microscopic examination and DNA ladder pattern was shown on electrophoresis. The average percentages of apoptosis measured by flow cytometric analysis were as follows;0.16 +/-0.08%in PBS, 0.23 +/-0.13%in 0.1 milligram/milliliter MMC, 0.50 +/-0.13%in 0.2 milligram/milliliter M M C , and 6.50 +/-1.57%in 0.4 milligram/milliliter MMC. Significant differences were shown in the percentage of apoptosis among the groups based on Kruskal-Wallis test[p=0.005]. Clinically relevant doses of MMC induces apoptosis in cultured keratocytes proportionally. This results suggest that MMC may modulate corneal wound healing process by accelerating the resolution phase of wound healing.
Apoptosis* ; DNA ; Electrophoresis ; Mitomycin* ; Propidium ; Wound Healing

BACKGROUND: The importance of the determination of cell viability has prompted the development of several assays of viability that utilize the exclusion of certain dyes by viable cell membranes. Recently, flow cytometry has been adapted to estimate cell viability by using fluorescent dye which is excluded by living cells on the basis of altered dead cell properties. OBJECTIVE: We have developed a flow Cytometric method for measuring cell viability after staining with propidium iodide (PI) and have compared it with the classical colorimetric method, MTT assay, which is currently widely used in cytotoxicity assays in the research field. METHODS: We performed flow cytometry and MTT assay for the comparison of the sensitivity of the assessment of cell viability. RESULTS: Decrease of cell viability was measured by flow cytometry with the addition of as little as 0.002% Triton-X 100 in comparison to MTT assay which could only reveal a similar decrease of cell viability with the new method to 0.008% Triton-X 100. CONCLUSION: Our results demonstrate this new method to be more sensitive and simple for the assessment of cell viability.
Cell Membrane ; Cell Survival* ; Coloring Agents ; Flow Cytometry ; Methods* ; Propidium*

BACKGROUND: Ultraviolet(UV) light is one of the injurious environmental agents which is known to lead to apoptosis of cells. However, studies on UVB-induced apoptosis of melanocytes are still lacking and there are some discrepancies between researchers. OBJECTIVE: Our purpose was to evaluate the characteristics of UVB-induced apoptosis of melanocytes and G361 cells. METHODS: Cultured normal human melanocytes and malignant melanoma cell lines (G361 cells) were analyzed by several detection methods including morphological examination of propidium iodide(PI) stained cells under fluorescence microscopy, quantitation of fragmented DNA, and flow cytometric analysis. RESULTS: Both melanocytes and G361 cells showed similar rate of apoptosis with gradual increment of UVB doses by the quantitation of fragmented DNA. However, flow cytometric analysis using scatter properties and PI stainability revealed that the melanocytes were more resistant to UVB than G361 cells. CONCLUSION: We suggest that melanocytes seem to be more resistant to UVB-induced injury than G361 cells. In addition, various methods for the detection of apoptosis might be necessary for its study. (Ann Dermatol 10:(3) 147152, 1998).
Apoptosis* ; Cell Line ; DNA ; Humans* ; Melanocytes* ; Melanoma ; Microscopy, Fluorescence ; Propidium

PURPOSE: To analyze the isolating pattern of slow cycling cells as putative limbal epithelial stem cells (PLESCs) using Hoechst exclusive cell sorting. METHODS: Rabbits were injected with 5-bromo-2-deoxyuridine (Brd U) 1 month prior to be sacrificed. After obtaining limbal tissues, fluorescence-activated cells were sorted on a Coulter EPICS 753 after they had been incubated with Hoechst 33342 and propidium iodide. Two different methods were applied to sort PLESCs. Side-population(Sp) cells were obtained using gates with dichroic mirror to detect low Hoechst blue and red after verapamil was treated. Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 BP filter. Brd U-retaining cells were counted and their sizes were evaluated in each gated sample to compare isolating pattern of PLESCs in each method. RESULTS: The percentages of Sp cells and of the Hoechst negative fraction were 0.96 +/- 0.79% and 16.01 +/- 13.60%, respectively(p=0.021). Homogeneity and density of the small cells were higher in Hoechst negative fraction than in Sp cells. The percentage of Brd U-retaining cells was 47.36 +/- 10.34% and 47.14 +/- 14.94% in Sp cells and Hoechst negative fraction, respectively(p>0.05), and they were 10 times higher than in non-Sp and Hoechst positive fraction(p=0.000). CONCLUSIONS: Hoechst negative exclusion without verapamil more efficiently isolated PLESCs than Sp did.
Cornea ; Epithelium ; Fluorescein* ; Propidium ; Rabbits ; Stem Cells* ; Verapamil

PURPOSE: Epileptic patients have a increasing tendency to develop seizure attack in high temperature. This finding suggests that high temperature may have an effect on neuronal hyperexcitability and injury of epileptic brain. Therefore, the influence of high temperature on normal and epileptic brain was studied in organotypic explant cultures of rat. METHODS: Fourteen days-in-vitro cultures from 8 day-old rat pups were perfused with standard aCSF bubbled with 95%/5% O2/CO2 in a microchamber. Stimulus train(0.3 sec, 60 Hz) was applied to Schaffer collaterals in CA3 and extracellular field potential was recorded in the CA1 pyramidal layer. At 36degrees C initially, AD was evoked. In high temperature(HT) group, the cultures were subjected to 39degrees C for a period of 8 min before the second stimulus train was applied. They were then restored to 36degrees C for 10 min. In normal temperature group, temperature was maintained at 36degrees C for the second stimulus train. The cultures were returned to the incubator and observed serially for neuronal damage. Intensity of propidium iodide fluorescence indicative of neuronal injury was quantitated by digital image analysis. The cultures on the same insert that were not stimulated served as the unstimulated groups. RESULTS: There was not a statistically significant difference in neuronal damage between the unstimulated high-temperature(HT) and normal-temperature(NT) group. In CA1 sector, % damage(mean+/-SEM) was 0.42+/-0.20 vs 0.27+/-0.05 at 24 hrs(HT vs NT group, n=16 each, P>0.05, Student t-test); 1.81+/-0.79 vs 1.43+/-0.27 at 48 hrs(P>0.05); 3.50+/-1.32 vs 3.35+/-0.56 at 72 hrs(P>0.05). In CA3 sector, % damage was 0.34+/-0.10 vs 0.20+/-0.03 at 24 hrs(P>0.05); 0.99+/-0.20 vs 0.83+/-0.23 at 48 hrs(P>0.05); 2.00+/-0.38% vs 2.26+/-0.35% at 72 hrs(P>0.05). Neuronal damage on AD induced cultures during febrile setting(n=16) was significantly higher than in nonfebrile setting(n=16). In CA1 sector, % damage was 6.63+/-2.56 vs 0.92+/-0.45 at 24 hrs(febrile setting vs nonfebrile setting, P= 0.036); 26.37+/-7.44 vs 4.99+/-2.23 at 48 hrs(P=0.010); 38.59+/-9.63 vs 6.48+/-2.30 at 72 hrs (P=0.003). In CA3 sector, % damage was 1.23+/-0.48 vs 3.91+/-2.37 at 24 hrs(P=0.277); 13.09+/-5.75 vs 5.93+/-3.27 at 48 hrs(P=0.288); 27.86+/-8.68 vs 7.54+/-3.74 at 72 hrs(P=0.04). CONCLUSION: At high temperature, seizures in epileptic brain may be more injurious than seizures in normal temperature.
Animals ; Brain ; Fluorescence ; Hippocampus* ; Humans ; Incubators ; Neurons* ; Propidium ; Rats* ; Seizures

PURPOSE: This study evaluated the extent of damage due to hypothermia in the mature and immature brain. METHODS: Hippocampal tissue cultures at 7 and 14 days in vitro (DIV) were used to represent the immature and mature brain, respectively. The cultures were exposed at 25degrees C for 0, 10, 30, and 60 minutes (n=30 in each subgroup). Propidium iodide fluorescent images were captured 24 and 48 hours after hypothermic injury. Damaged areas of the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis. RESULTS: At 7 DIV, the tissues exposed to cold injury for 60 minutes showed increased damage in CA1 (P<0.001) and CA3 (P=0.005) compared to the control group at 48 hours. Increased damage to DG was observed at 24 (P=0.008) and 48 hours (P=0.011). The 14 DIV tissues did not demonstrate any significant differences compared with the control group, except for the tissues exposed for 30 minutes in which DG showed less damage at 48 hours than the control group (P=0.048). In tissues at 7 DIV, CA1 (P=0.040) and DG (P=0.013) showed differences in the duration of cold exposure. CONCLUSION: The immature brain is more vulnerable to hypothermic injury than the mature brain.
Animals ; Brain ; Dentate Gyrus ; Hippocampus ; Hypothermia, Induced* ; Neurons* ; Propidium ; Rats*

PURPOSE: The aim of this study was to investigate the potential effects of mild hypoxia in the mature and immature brain. METHODS: We prepared organotypic slice cultures of the hippocampus and used hippocampal tissue cultures at 7 and 14 days in vitro (DIV) to represent the immature and mature brain, respectively. Tissue cultures were exposed to 10% oxygen for 60 minutes. Twenty-four hours after this hypoxic insult, propidium iodide fluorescence images were obtained, and the damaged areas in the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis. RESULTS: In the 7-DIV group compared to control tissue, hypoxia-exposed tissue showed decreased damage in two regions (CA1: 5.59%+/-2.99% vs. 4.80%+/-1.37%, P=0.900; DG: 33.88%+/-12.53% vs. 15.98%+/-2.37%, P=0.166), but this decrease was not statistically significant. In the 14-DIV group, hypoxia-exposed tissue showed decreased damage compared to control tissues; this decrease was not significant in the CA3 (24.51%+/-6.05% vs. 18.31%+/-3.28%, P=0.373) or DG (15.72%+/-3.47% vs. 9.91%+/-2.11%, P=0.134), but was significant in the CA1 (50.91%+/-5.90% vs. 32.30%+/-3.34%, P=0.004). CONCLUSION: Although only CA1 tissues cultured for 14 DIV showed significantly less damage after exposure to hypoxia, the other tissues examined in this study showed a tendency towards less damage after hypoxic exposure. Therefore, mild hypoxia might play a protective role in the brain.
Anoxia* ; Brain ; Dentate Gyrus ; Fluorescence ; Hippocampus ; Neuroprotective Agents* ; Oxygen ; Propidium

PURPOSE: The use of an autogenous fat graft has become a common procedure in plastic surgery. However, questions remain concerning on the viability of fat cells and preservation method of aspirated fat. The purpose of this study is to examine the viability of fat cells stored at -20degrees C in the freezer for 1 year after harvest from abdominal liposuction. METHODS: Eighteen adults(aged from 24 to 65 years, 16 female and 2 male) were selected for this study. Harvested aspirated fat tissues were obtained by suction-assisted lipectomy and frozen at -20degrees C commercial refrigerator for one year(average 12.5 months). The viability of fat cells in specimens were measured after thawing. The numbers of viable cells were measured on a fluorescence microscope after staining with fluorescein diacetate and propidium iodide. GPDH(Glycerol-3-phosphate dehydrogenase) activity was measured. Cell culture was done for 3 weeks. RESULTS: There were no viable cells under the fluorescence microscope, no detectable GPDH activity, and no cultured cells. CONCLUSION: These findings suggest that aspirated fat after frozen storage for one year at -20degrees C freezer is inadequate to reuse.
Adipocytes ; Cell Culture Techniques ; Cryopreservation ; Female ; Fluorescein ; Fluoresceins ; Fluorescence ; Humans ; Lipectomy ; Propidium ; Surgery, Plastic ; Transplants