To begin, label two new fax tubes as mixed tubes one and two. Prepare the cell and antibody suspension as per the given table for flow cytometric analysis. After incubating the tubes for 30 minutes, wash each tube two times with one milliliter of staining buffer.
Centrifuge the vortexed tubes at 200 G for 10 minutes at room temperature. Then re-suspend the pellet in 500 microliters of staining buffer. Coat the wells of a 48 well plate with 200 to 500 microliters of FBS, and keep for one hour.
Then pipette 200 to 500 microliters of 10%MSC media after removing the residual FBS. To prepare the samples for single cell sorting, pipette one milliliter of FBS into two new fax tubes. Roll the tubes to create an even coat of the FBS on the tube's inner surface.
Prepare the cell and antibody suspension in mixed tubes one and two, as per the table, for the sorting experiment. Then incubate the tubes in the dark for 30 minutes at room temperature. After washing with staining buffer, centrifuge the tubes at 200 G for 10 minutes at room temperature.
Then re-suspend the pellet in 500 microliters of staining buffer and incubate for 15 minutes in five microliters of DAPI before sorting. To set up the compensation matrix, use the single stain compensation tubes. Click on experiment from the toolbar in the instrument software.
Then click on compensation setup, followed by create compensation controls. Next, click on unstained tube, followed by parameters in the cytometer dialogue box. Adjust the voltages by editing the values for each parameter.
Click on load in the acquisition dashboard and then click acquire. Drag the P-1 gate to the cell population and apply it across all compensation controls to set the voltage and negative gate for each parameter. Now load the single stain compensation tubes individually.
Record the data and save it. Select the current experiment, right click on it, and select calculate compensation values, followed by link and save. Run the mixed tube one and record 10, 000 cells.
Set the gates on the population of interest to be sorted with the proper gating strategy. Next, load a collection plate into the instrument and set a target cell number, ranging from 2, 500 to 5, 000 cells per well. Then select the single cell sort purity mask.
Collect the sorted cell populations in a collection device, keeping them on ice for the duration of the sorting experiment. Finally, shift the plates into a 5%carbon dioxide incubator to maintain the cultures at 37 degrees Celsius. The multi-parametric flow cytometry assays plots showed no expression of the FITC isotype of CD 90 FITC antibodies.
Positive expression of the markers CD-90 and CD-73 and negative expression of CD-45 are comparable with that of their unstained and FMO controls. The immuno phenotypes showed the marker distribution from one of the early passages with the ISCT recommended markers and CD-44, determining the parent populations as MSCs. The late passage sheds were used for single cell sorting of high and dim expressors.
The post sorted cells collected in the 48 well plate showed adherence and proliferation, like their parent population. The post sorted cells differentiated in the presence of adipogenic growth factors.
