Optimal amounts of http://www.selleckchem.com/products/ABT-263.html antibodies were determined earlier.2.4. MeasurementsMeasurements were carried out using a FACS Calibur flow cytometer (Becton Dickinson) on the day of the staining, collecting 5 �� 104cells/tube. CellQuest-Pro software program (Becton Dickinson) was used for analysis. Detected curves (histograms) were separated based on their fluorescence intensity; control peak derived from ��isotype�� control antibodies, while positive histogram peaks derived from specific antibodies. Quantitative percentage of cells with the investigated properties was calculated by the software after separated the histograms with markers (see Figure 1, M1 marker).Figure 1CD59 expression of amniotic cells. Count of the CD59+ cells versus the intensity of cell staining is presented.

(a): fresh, (b): 1 month, (c): 4 months, (d): 14 months freezing time. The intervals signed by M1 marker mark out the CD59-positive cells. …2.5. Statistical AnalysisKruskal-Wallis nonparametric analysis of variance was performed to compare the percentage of the CD59+ cells of the freshly prepared and cryopreserved samples.3. Results3.1. Effect of Cryopreservation on the CD59 Expression of the Amniotic MembranesCD59-positive cells could be detected in all investigated amniotic membrane samples. When we examined the same fresh sample before and after 15 minutes freezing, differences could not been detected in the fluorescence intensities (data not shown). The percentage of the CD59+ cells varied among both the freshly prepared and cryopreserved (1 to 14 months) samples (46�C94% and 39�C98%, resp.).

There was, however, no statistically significant difference in the proportion of the CD59+ cells between the fresh and preserved samples (Table 2).Table 2Rate of CD59 positive cells in amniotic membrane pieces processed after different freezing times. The rates are given in percent, as mean �� standard deviation. Markedly no relation can be discovered in the rate of CD59-positive cells and the freezing …The percentage of CD59+ cells was variable even among different pieces of the same placenta at the same preservation time. The variation coefficient of the measurement in fresh placentas was 22.9%.3.2. Expression of CD59 on Different Cell Types of the Amniotic Membranes The detected CD59 histograms frequently show a bimodal (2 peak) distribution (Figure 1). Brefeldin_A Amniotic membranes contain different cell types, most of them are epithelial cells and mesenchymal cells. We supposed that the bimodal histograms were caused by the different expression levels of CD59 on different amniotic cell types. Using multicolor flow cytometric measurements we compared the cell surface expression of CD59 of epithelial and mesenchymal cells. CD59 were found to be expressed in both types of cells.