Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers

Begin by adjusting the concentration of thawed murine adipose-derived stem cell suspension using PBS with 5%fetal bovine serum. Aliquot 100 microliters of the suspension containing 100, 000 cells into a 1.5 milliliter centrifuge tube. Add appropriate amounts of anti-CD105 AF594 and anti-CD31 AF680 as per the manufacturer's instructions.

Incubate for 20 minutes in the dark at four degrees Celsius. Remove excess stain by washing with PBS/FBS and centrifuge at 250 x g for five minutes. Resuspend the cells in 300 microliters of 1%formalin.

Create a dot plot graph by clicking on the polygon symbol to select the subpopulation of interest and exclude the debris areas using FSC-A versus SSC-A gating. Next, click on the polygon symbol to generate a new dot plot and exclude the duplexes and multiples cells using SSC-H versus SSC-A followed by FSC-H and FSC-A. Use Y610-mCHERRY for the AF594 and the R660 APC-A detectors for the AF680 fluorophores, respectively.

Click on the quadrant symbol and set each detector's threshold for autofluorescence cells. Select samples labeled with CD105 and positive population to be shown on Q1LR. Next, choose samples labeled with CD31 and positive population to be shown on Q1LR.

Immunophenotyping showed that 98.7%of the cell population was positive for CD105, while only 5.88%expressed the CD31 marker.