Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Cytotoxicity of newly developed pozzolan cement and other root-end filling materials on human periodontal ligament cell

OBJECTIVES: The purpose of this study was to evaluate in vitro cytotoxicity of the pozzolan cement and other root-end filling materials using human periodontal ligament cell. MATERIALS AND METHODS: Endocem (Maruchi), white ProRoot MTA (Dentsply), white Angelus MTA (Angelus), and Super EBA (Bosworth Co.) were tested after set completely in an incubator at 37degrees C for 7 days, Endocem was tested in two ways: 1) immediately after mixing (fresh specimens) and 2) after setting completely like other experimental materials. The methods for assessment included light microscopic examination, cell counting and WST-1 assay on human periodontal ligament cell. RESULTS: In the results of microscopic examination and cell counting, Super EBA showed significantly lower viable cell than any other groups (p < 0.05). As the results of WST-1 assay, compared with untreated control group, there was no significant cell viability of the Endocem group. However, the fresh mixed Endocem group had significantly less cell viability. The cells exposed to ProRoot MTA and Angelus MTA showed the highest viability, whereas the cells exposed to Super EBA displayed the lowest viability (p < 0.05). CONCLUSIONS: The cytotoxicity of the pozzolan cement (Endocem) was comparable with ProRoot MTA and Angelus MTA. Considering the difficult manipulation and long setting time of ProRoot MTA and Angelus MTA, Endocem can be used as the alternative of retrofilling material.

The evaluation of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure / 대한치과보존학회지

The purpose of this study was to evaluate the viability of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure by means of MTT assay, WST-1 assay. 12 teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both side of the first and second maxillary molars were extracted as atraumatically as possible under tiletamine anesthesia. The experimental groups were group 1 (Immediate extraction), group 2 (Slow freezing under pressure of 3 MPa), group 3 (Slow freezing under pressure of 2 MPa), group 4 (Slow freezing under no additional pressure), group 5 (Rapid freezing in liquid nitrogen under pressure of 2 MPa), group 6 (Rapid freezing in liquid nitrogen under no additional pressure), group 7 (low-temperature preservation at 0degrees C under pressure of 2 MPa), group 8 (low-temperature preservation at 0degrees C under no additional pressure), group 9 (low-temperature preservation at -5degrees C under pressure of 90 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in 37degrees C water bath, then MTT assay, WST-1 assay were processed. One way ANOVA and Tukey HSD method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 7 (0degrees C/2 MPa) showed higher viability of periodontal ligament cells than other group (2-6, 8) and this was statistically significant (p < 0.05), but showed lower viability than group 1, immediate extraction group (no statistical significance). By the results of this study, low-temperature preservation at 0degrees C under pressure of 2 MPa suggest the possibility for long term preservation of teeth.

The efficacy of programmed cryo-preservation under pressure in rat periodontal ligament cells / 대한치과보존학회지

The purpose of this study was to evaluate the viability of periodontal ligament cells in rat teeth using slow cryo-preservation method under pressure by means of MTT assay and WST-1 assay. Eighteen teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both sides of the first and second maxillary molars were extracted as atraumatically as possible under Tiletamine anesthesia. The experimental groups were group 1 (Immediate control), group 2 (Cold preservation at 4degrees C for 1 week), group 3 (Slow freezing), group 4 (Slow freezing under pressure of 3 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in 37degrees C water bath, then MTT assay and WST-1 assay were processed. One way ANOVA and Tukey method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 4 showed significantly higher viability of periodontal ligament cells than group 2 and 3 (p < 0.05), but showed lower viability than immediate control group. By the results of this study, slow cryo-preservation method under pressure suggests the possibility for long term cryo-preservation of the teeth.

Comparison of viability of oral epithelial cells stored by different freezing methods / 대한치과보존학회지

This study examined the influence of the storage methods on the viability of oral epithelial cells using conventional cell freezing storage, slow freezing preservation, rapid freezing preservation, and slow freezing preservation with a pressure of 2 Mpa or 3 Mpa. The cell viability was evaluated by cell counting, WST-1 and the clonogenic capacity after 6 days of freezing storage. After 6 days, the frozen cells were thawed rapidly, and the cell counting, WST-1, and clonogenic capacity values were measured and compared. 1. The results from cell counting demonstrated that conventional cryopreservation, slow freezing under a 2 Mpa pressure and slow freezing under a 3 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05). 2. The results from the optical density by WST-1 demonstrated that slow freezing under a 2 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05). 3. The clonogenic capacity demonstrated that slow freezing under a 2 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05).

The Effects of Peroxiredoxin III on Human HeLa Cell Proliferation

BACKGROUND: Peroxidases (Prx) of the peroxiredoxin family reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol respectively. Hydrogen peroxide is implicated as an intracellular messenger in various cellular responses such as proliferation and differentiation. And Prx I activity is regulated by Cdc-2 mediated phosphorylation. This work was undertaken to investigate the proliferation role of peroxiredoxin III as a member of Prx family in Prx III overexpressed HeLa cell line. METHODS: To provide further evidence of proliferation, we selected Prx III stably expressed HeLa Tet-off cell lines. Cell proliferation was examined by using proliferation reagent WST-1 in the presence or absence of doxycycline. Prx III, 2-cys Prx enzymes exist as homodimer. The activation of Prx III heterodimer with induced and endogenous Prx III was examined by immunoprecipitation. RESULTS: Immunoprecipitation analysis of the induced and endogenous Prx III with anti-myc showed that the induced wild type (WT) and dominant negative (DN) Prx III from HeLa Prx III Tet-off stable cell heterodimerized with endogenous Prx III each other. And the expression level of induced Prx III was examined after addition of doxycycline. By 72 hr, the expression level of induced Prx III was diminished gradually and the half-life of the induced wild type Prx III was approximately 17 hr. The proliferation experiment demonstrated that the relative proliferation value of induced and endogenous WT Prx III stable cell has no changes but the DN Prx III induced HeLa Tet-off stable cells were lower than endogenous Prx III. CONCLUSION: In conclusion, the HeLa dominant negative Prx III Tet-off stable cells were decreased the proliferation.