To begin, stain the murine bone marrow cells with Hoechst 33342. For flow cytometry, launch the required software, then select the QC standardization. Insert the quality control or QC FluoroSphere sample tube into the tube holder before selecting start to begin the QC procedure.
To create a new experiment, click on New Experiment in the File menu and specify the file path and save the experiment. Then select Set Channel in the Settings menu. Select the channel signal checkbox, and add the reagent name in the label column.
Next, click pseudo color plots icon in the plot area to create plots. Click add tube in the test tube screen to create new sample tubes and change their names. Then select Run to load the sample, view the plots and establish the gates.
Adjust the gain and threshold settings and select Record to save the data. To design the gate setting logic, click the x-axis to select FSEW, and click the y-axis to select FSEA. Select Polygon Gate to draw gate A to circle the individual cell and exclude adherent cells.
For the second plot, click the x-axis to select FSEA, and click the y-axis to select SSEA. Select Polygon Gate to draw gate B to separate non-fragmentary cells and exclude cellular debris. For the third plot, click the x-axis to select PIA, and click the y-axis to select SSEA.
After drawing the gate B, select live cells exhibiting negative propidium iodide and draw gate C to obtain live cells. For the fourth two-dimensional plot, click the x-axis to select Hoechst Red, and click the y-axis to select Hoechst Blue. Right-click the plot and select Property from the dropdown menu.
Select linear format for both the x-axis and y-axis and draw the gate for side population cells. Use the 355 nanometer and 405 nanometer lasers simultaneously to detect both the control cells and the experimental cells. Acquire fluorescent signals at the 690 by 50 and 450 by 50 nanometer channels corresponding to the two laser.
Observe the effective stimulation of Hoechst 33342 dye with 355 and 405 nanometer lasers to visualize the clear side population cells. For the same samples, use the 375 nanometer and 405 nanometer lasers for cell detection. The 355 nanometer laser effectively excited the Hoechst 33342 dye, resulting in clear observation of SP cells of bone marrow.
The SP cells of the same samples detected by the 355 nanometer laser and the 405 nanometer laser were found to be essentially identical. Both 375 nanometer and 405 nanometer lasers effectively excited Hoechst 33342 to obtain SP cells.
