We use the Hoechst dye population method to detect tumor initiating cells. In this paper, we're trying to find alternative approaches for Hoechst excitation. We found that the high power 375 nanometer and 405 nanometer lasers can serve as feasible alternatives to the traditional 355 nanometer laser.
Conventionally, the Hoechst dye is excited with a UV laser at 355 nanometer. Due to the high cost of the UV lasers, they're not commonly equipped in most flow cytometry models. Our protocol expands a range of the lasers that can be used to detect Hoechst staining.
For researchers focusing on normal and malignant stem cells, more models of flow cytometry can be used to detect side population cells. To begin, obtain bone marrow cells from the mouse and add five micrograms per milliliter Hoechst 33342 to two milliliters of cell suspension. Prepare the negative control tube as well.
Incubate in a water bath at 37 degrees Celsius for 90 minutes with gentle agitation every 30 minutes After incubation, cool the cells on ice for five minutes and then centrifuge at 250 G for five minutes at four degrees Celsius. Resuspend the cells in a cold running solution containing Hank's Balanced Salt Solution with 2%FBS. Centrifuge again at four degrees Celsius for five minutes.
Next, resuspend the resulting cell pellet in 500 microliters of running solution per sample. Prior to flow cytometry analysis, supplement cells with two micrograms per milliliter of propidium iodide and place them on ice for approximately five minutes. To begin, stain the mirroring bone marrow cells with Hoechst 33342.
For flow cytometry, launch the required software. Then select in the QC standardization. Insert the quality control or QC fluoro sphere 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 SSCA. 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 SSCA. 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 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.
