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As the study is cross-sectional, it is not viable to exclude "the chicken or the egg" causality dilemma.
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Cell numbers and viabilities were assessed using a hemocytometer based on the ability of the viable cells to exclude trypan blue.
Integrity of the plasma membrane is shown by the ability of a viable cell to exclude the dye, whereas the dye will diffuse passively into sperm cells with damaged plasma membranes [ 22].
The ability to exclude viable dyes such as trypan blue, propidium iodide (PI) or 7-aminoactinomycin D (7AAD) is a property of cells that have an intact plasma membrane.
To exclude any viable M. synoviae, the membrane fraction was treated with phosphate buffered saline (PBS) containing 0.5 mg gentamicin/mL for 4 h at 38°C.
To exclude non-viable cells in our experiments, all samples were stained with propidium iodide (PI).
Cell suspensions were analyzed by a flow-cytometer (Epics XL analyzer, Coulter Corporation, Miami, FL) after addition of 5l of a 1mg/ml solution of propidium iodide to exclude non-viable cells.
Selection for PE-conjugated CD31+ endothelial cells was carried out by first gating on larger cells within a SSC-A and FSC-A dot plot to exclude non-viable cells and debris.
SYTOX green (Invitrogen) was used to exclude non-viable cells.
Gates were set to exclude non-viable cells and cell debris based on light scattering properties.
After 24 h, they were harvested by trypsinization and subjected to FACS analysis using a CyAN cytometer (Beckman-Coulter) and standard gating procedures to exclude non-viable cells.
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