thawing processes. Perform the characterization by staining the
nuclei with DAPI stain for visualization of the polyploidy of MKs
with fluorescence microscopy.
1. Concentrate MKs from a suspension by centrifugation
(120 Â g, 10 min), and resuspend in DPBS. For recovery of
cells after cryopreservation, refer to the corresponding thawing
procedure section.
2. Work with 1 Â 10
4 cells.
3. Transfer 100 μL of DPBS MK cell suspension in a 5 mL
test tube.
4. Stain cells with 5 μL of MK surface marker-specific FITCconjugated antibody (e.g., anti-CD61-FITC).
5. Gently vortex the tube and incubate for 15 min at room
temperature in the dark.
6. Wash cells with DPBS by centrifugation (120 Â g, 10 min).
7. Aspirate supernatant. Permeabilize and fix the MK cell membranes by adding 200 μL of Cytofix/Cytoperm™ fixation/
permeabilization solution for 20 min at room temperature in
the absence of light.
8. Wash cells with DPBS by centrifugation (120 Â g, 10 min).
9. Resuspend the pellet in 200 μL of 1:1000 dilution of DAPI in
DPBS, and incubate at room temperature in the absence of
light for 3 min.
10. Wash cells with DPBS by centrifugation (120 Â g, 10 min).
11. Resuspend the pellet in 500 μL of DPBS, and transfer into
1 well of a 24-well suspension culture plate.
12. Allow cells to sediment by gravity for 5 min at 37
C.
13. Analyze by fluorescent microscopy using overlays of the DAPI
and the GFP channel at 40Â magnification. Identify the cells
with membranes stained in green (due to the MK-specific
staining with anti CD61-FITC) and multiple nuclei (DAPI
staining of polyploid nuclei). Figure 4 shows polyploidy of
MKs analyzed with nuclear DAPI staining by fluorescence
microscopy before cryopreservation as well as after cryopreservation with 7.5% DMSO.
3.8 Characterization
of iPSC-Derived MKs
for Polyploidy
with Flow Cytometry
Quantitative evaluation of polyploidy of iPSC-derived MK can be
performed by analysis of DNA content in the cells with propidium
iodide staining. Since propidium iodide under physiological conditions cannot penetrate through an intact membrane, the cells have
to be permeabilized. Signal peaks indicate ploidy of analyzed cell
populations.
Cryopreservation of iPSC-Derived Megakaryocytes
549
nuclei with DAPI stain for visualization of the polyploidy of MKs
with fluorescence microscopy.
1. Concentrate MKs from a suspension by centrifugation
(120 Â g, 10 min), and resuspend in DPBS. For recovery of
cells after cryopreservation, refer to the corresponding thawing
procedure section.
2. Work with 1 Â 10
4 cells.
3. Transfer 100 μL of DPBS MK cell suspension in a 5 mL
test tube.
4. Stain cells with 5 μL of MK surface marker-specific FITCconjugated antibody (e.g., anti-CD61-FITC).
5. Gently vortex the tube and incubate for 15 min at room
temperature in the dark.
6. Wash cells with DPBS by centrifugation (120 Â g, 10 min).
7. Aspirate supernatant. Permeabilize and fix the MK cell membranes by adding 200 μL of Cytofix/Cytoperm™ fixation/
permeabilization solution for 20 min at room temperature in
the absence of light.
8. Wash cells with DPBS by centrifugation (120 Â g, 10 min).
9. Resuspend the pellet in 200 μL of 1:1000 dilution of DAPI in
DPBS, and incubate at room temperature in the absence of
light for 3 min.
10. Wash cells with DPBS by centrifugation (120 Â g, 10 min).
11. Resuspend the pellet in 500 μL of DPBS, and transfer into
1 well of a 24-well suspension culture plate.
12. Allow cells to sediment by gravity for 5 min at 37
C.
13. Analyze by fluorescent microscopy using overlays of the DAPI
and the GFP channel at 40Â magnification. Identify the cells
with membranes stained in green (due to the MK-specific
staining with anti CD61-FITC) and multiple nuclei (DAPI
staining of polyploid nuclei). Figure 4 shows polyploidy of
MKs analyzed with nuclear DAPI staining by fluorescence
microscopy before cryopreservation as well as after cryopreservation with 7.5% DMSO.
3.8 Characterization
of iPSC-Derived MKs
for Polyploidy
with Flow Cytometry
Quantitative evaluation of polyploidy of iPSC-derived MK can be
performed by analysis of DNA content in the cells with propidium
iodide staining. Since propidium iodide under physiological conditions cannot penetrate through an intact membrane, the cells have
to be permeabilized. Signal peaks indicate ploidy of analyzed cell
populations.
Cryopreservation of iPSC-Derived Megakaryocytes
549