9. Important: it is always necessary to compare results of trypan
blue exclusion test analysis for the survival of MKs after cryopreservation procedures with non-cryopreserved control. Such
control should be a general rule for all cell types, but is especially important for the MKs due to natural remodeling events
that occur in the membrane and in the cytoplasm of this cell
type, which is necessary for PLT production [20].
10. Normalize results to non-cryopreserved MK control.
3.5 Evaluation
of Survival
with Distinguishing
of Apoptotic Processes
in iPSC-Derived MKs
After Cryopreservation
with Annexin V
and Propidium Iodide
Use APC Annexin V Apoptosis Detection Kit with PI for the
analysis of necrotic and apoptotic mechanisms, which can be triggered in MKs as a result of cryopreservation procedures (the protocol is provided for application of APC Annexin V Apoptosis
Detection Kit with PI, BioLegend, CA, USA).
1. For recovery of cells after cryopreservation, refer to the
corresponding thawing procedure section.
2. Concentrate MKs from a suspension by centrifugation
(120 Â g, 10 min), and resuspend in 1 mL of cold (4
C)
BioLegend’s Cell Staining Buffer.
3. Wash cells twice with cold BioLegend’s Cell Staining Buffer
(120 Â g, 10 min), and then resuspend the pellet in 100 μL of
Annexin V Binding Buffer.
4. Transfer 100 μL of cell suspension in a 5 mL test tube.
5. Stain cells with 5 μL of MK marker-specific fluorochromeconjugated antibody to distinguish MKs from the whole cell
population. Make sure that antibody fluorochrome spectrum
does not overlap with APC channel of Annexin V or with PE
channel of propidium iodide (e.g., use APC-Cy7-conjugated
anti-CD41 antibody).
6. Add 5 μL of APC Annexin V.
7. Add 10 μL of propidium iodide solution.
8. Gently vortex the tube and incubate for 15 min at room
temperature in the dark.
9. Add 400 μL of Annexin V Binding Buffer to each tube.
10. Analyze by flow cytometry with at least 10,000 events per
measurement. First, gate the cells according to forward
scatter vs. side scatter (FSC vs. SSC) parameters, and then
analyze MK-specific population. The MKs that are stained
only with Annexin V are at the early stage of apoptosis. The
MKs that are stained with PI are necrotic or at a late apoptotic
stage. Figure 2 shows typical dot plots of flow cytometry
analysis of iPSC-derived MK population before cryopreservation as well as after cryopreservation with 7.5% DMSO.
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Denys Pogozhykh et al.
blue exclusion test analysis for the survival of MKs after cryopreservation procedures with non-cryopreserved control. Such
control should be a general rule for all cell types, but is especially important for the MKs due to natural remodeling events
that occur in the membrane and in the cytoplasm of this cell
type, which is necessary for PLT production [20].
10. Normalize results to non-cryopreserved MK control.
3.5 Evaluation
of Survival
with Distinguishing
of Apoptotic Processes
in iPSC-Derived MKs
After Cryopreservation
with Annexin V
and Propidium Iodide
Use APC Annexin V Apoptosis Detection Kit with PI for the
analysis of necrotic and apoptotic mechanisms, which can be triggered in MKs as a result of cryopreservation procedures (the protocol is provided for application of APC Annexin V Apoptosis
Detection Kit with PI, BioLegend, CA, USA).
1. For recovery of cells after cryopreservation, refer to the
corresponding thawing procedure section.
2. Concentrate MKs from a suspension by centrifugation
(120 Â g, 10 min), and resuspend in 1 mL of cold (4
C)
BioLegend’s Cell Staining Buffer.
3. Wash cells twice with cold BioLegend’s Cell Staining Buffer
(120 Â g, 10 min), and then resuspend the pellet in 100 μL of
Annexin V Binding Buffer.
4. Transfer 100 μL of cell suspension in a 5 mL test tube.
5. Stain cells with 5 μL of MK marker-specific fluorochromeconjugated antibody to distinguish MKs from the whole cell
population. Make sure that antibody fluorochrome spectrum
does not overlap with APC channel of Annexin V or with PE
channel of propidium iodide (e.g., use APC-Cy7-conjugated
anti-CD41 antibody).
6. Add 5 μL of APC Annexin V.
7. Add 10 μL of propidium iodide solution.
8. Gently vortex the tube and incubate for 15 min at room
temperature in the dark.
9. Add 400 μL of Annexin V Binding Buffer to each tube.
10. Analyze by flow cytometry with at least 10,000 events per
measurement. First, gate the cells according to forward
scatter vs. side scatter (FSC vs. SSC) parameters, and then
analyze MK-specific population. The MKs that are stained
only with Annexin V are at the early stage of apoptosis. The
MKs that are stained with PI are necrotic or at a late apoptotic
stage. Figure 2 shows typical dot plots of flow cytometry
analysis of iPSC-derived MK population before cryopreservation as well as after cryopreservation with 7.5% DMSO.
546
Denys Pogozhykh et al.