This cell line was shown to posses the same ster oidogenic capabilities as primary human granulosa cells

Signal detection was done using x ray films and care was taken to expose the CM BSA and CM PA arrays equally. CM were either heated at 95 C for 15 or treated INK 128 価格 with 100ugmL proteinase K for 2 h, then heat inactivated as above to avoid damage of macro phages by proteinase K. Immunofluorescence staining and cell shape score All reagents were diluted in PBS supplemented with cal cium and magnesium, and cells were washed with PBS between each staining step. Cells cultured on glass co verslips were fixed with 3% paraformaldehyde for 30 min and incubated sequentially with 0. 1% Triton X100 for 10 min, 5% BSA for 30 min, and rhodamine phalloidin for 45 min. Nuclei were counterstained with 1 ugml DAPI for 5 min. Coverslips were then mounted onto glass slides and stored at 4 C until analysis.<br><br> Fluorescence images were captured on a spin ning disk confocal microscope using a 40X air objective. For an analysis unbiased by cell shape, fields were selected by viewing nuclear staining. Subsequent, rhodamine fluorescence was captured in x, y and z axes to detect actin within KU-57788 価格 the whole cell volume. At least 300 cells were used for analysis per condition. Shape quantification For each field, extended focus images were created and image analysis was per formed using ImageJ software. For each field, the image was transformed in 8 bits and a median filter was applied to approximate the distribution of staining intensity. Binary image masks were then created using an automatic threshold to create an image including all fluorescence data above back ground.<br><br> Detection of surface area was done using of the Analyze Particles option with a threshold of 100 pixels in order to exclude all small dots of fluorescence outside of cells. Quantitative fluorescence data were exported from ImageJ for further analysis and pre sentation. Area results given in pixel were then trans formed in um2 according Lonafarnib 193275-84-2 to the scale. Additionally, the elongated vs. round phenotype was determined by ma nual counting of the cells and eye determination of the shape. This quantification was done by a sample identity blinded experimenter to avoid subjective bias. Adhesion assay RAW macrophages were pre stained with calcein AM for 20 min and then centrifuged, washed and re suspended in each test CM from the muscle cell cultures. After 30 min, macrophages were seeded onto a 24 well plate and allowed to adhere for 15 min.<br><br> Me dium was then removed, cells were washed and the remaining adherent cells were lysed in 1% Triton X100. Fluorescence at excitationemission of 495515 nm was proportional to the number of adhered cells. Statistical analysis Statistical analysis was performed using GraphPad Prism software. Two groups comparison was performed using Students paired t test with Welsh correction for inequality of variances as necessary. Results of time courses were analyzed by two way analysis of variance followed as appropriate by Bonferroni post tests. One way ANOVA was used to test differences between groups. Data are presented as means SD or SEM, and statistical significance was set at P 0. 05.