Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

This protocol shows two distinct ways of detecting NKG2DLs on the surface of AML cells. The technique provides a fast, user-friendly staining method to detect all known and possibly unknown NKG2DLs. This method allows for separation of leukemic stem cells from bulk AML cells, which makes it possible to further characterize these cells.

Begin by thawing the biotin and the NKG2D fusion protein tubes at room temperature. Quickly spin down the NKG2D FC powder, then add 500 microliters of PBS to reconstitute the powder and mix thoroughly using a P-1000 micropipette. Add 100 microliters of the NKG2DL fusion protein to a biotin tube to obtain a final concentration of 10 micrograms per milliliter, and mix the solution thoroughly with a P-100 micropipette.

To thaw the AML cells, remove the cryovial containing primary AML from the liquid nitrogen storage, and immediately place it in a 37 degrees Celsius water bath. Gently move the tube back and forth in the water, allowing the contents of the vial to thaw until there is only a small ice crystal left. Immediately transfer the thawed cells into the medium containing tube and rinse the vial using one milliliter of medium.

Centrifuge the cells at 300 times G for 10 minutes and discard the supernatant without disturbing the pellet. Wash the cells with five milliliters of RPMI medium containing 10%FCS and repeat the centrifugation. Resuspend the cell pellet with staining buffer to a final concentration of 0.5 times 10 to the seventh cells per milliliter.

Then transfer 100 microliters of the cell suspension to a cell culture 96 well U-bottom plate and centrifuge the plate at 300 times G for 10 minutes. Discard the supernatant without disturbing the pellet. Prepare a master mix of biotinylated NKG2D fusion protein so that the cells are resuspended in a final volume of 50 microliters per well with a final NKG2D concentration of 10 micrograms per milliliter per well.

Add the prepared master mix and resuspend the cell pellets with a 200 microliter pipette. Incubate and centrifuge the plate as described in the text manuscript. Prepare a master mix using streptavidin PE so that the cells are resuspended in a final volume of 50 microliters.

Add the master mix to the cells and resuspend the pellets with a 300 microliter multichannel pipette. After centrifuging the plate and discarding the supernatant, use a 300 microliter multi-channel micropipette to resuspend the cell pellets in 200 microliters of staining buffer plus 7-AAD. Then analyze the cells using a flow cytometry device.

The analyzed AML samples are positive for CD34 and NKG2DL, but negative subpopulations also exist, with four different populations in total. The typical gating strategy starts with the selection of the main population of cells via their FSC and SSC. Doublets and dead cells are excluded in the downstream analysis.

The gates are adjusted using FMO controls to ensure proper identification of positive cells. The fluorescence intensities of cells positive for CD34 versus NKG2DL are highlighted here. AML cells that are positive for CD34 show a lower surface expression of NKG2DL, indicating that NKG2DL expression is associated with lack of stemness.

Three primary AML samples demonstrated 19.8, 49.8, and 89.4%positive events for the fusion protein staining versus 20.4, 50.4, and 90.6%for the pooled anti-NKG2DL antibody staining. On the other hand, single ligand staining showed a range of positive events up to 92%with the percentages varying based on the ligand. When attempting this method, it is very important to be quick when thawing the primary AML cells, because they are fragile and can die easily during this step.

After performing this protocol, a cell sort based on the NKG2DL signal can be performed to further investigate the populations.