To begin, centrifuge the trypsinised mesenchymal stem cells at 300 g for six minutes to obtain the cell pellet. Next, resuspend the pellet in one milliliter of MSC media before cell counting. Centrifuge the cell suspension again, then resuspend the pellet in an appropriate volume of the staining buffer after discarding the supernatant.
For the preparation of compensation controls, label seven fax tubes as unstained, DAPI, V450, FITC, PE, PerCP-Cy5.5, and APC. Next, prepare the single stain tubes for compensation and incubate them in the dark for 30 minutes. Following incubation, wash the cells in each tube two times and the beads one time with one milliliter of staining buffer.
Next, centrifuge the vortexed tubes at 200 g for 10 minutes at room temperature. After discarding the supernatant, resuspend the pellet in 500 microliters of staining buffer. Incubate the DAPI tube in a 60 degree Celsius water bath for five minutes followed by a 15-minute incubation on ice to induce the inclusion of the dye.
Add five microliters of DAPI to the suspension and incubate until acquisition. Prepare the cell and antibody suspension in five labeled fax tubes as per the table. Gently vortex the tubes before incubation.
Then, wash each tube two times with one milliliter of the staining buffer. Centrifuge the vortex tube at 200 g for 10 minutes at room temperature, then resuspend the remaining pellet in 500 microliters of staining buffer.
