Vancouver, Canada), supplemented with cytokines and growth
factors for terminal stage of differentiation of iPSCs into MKs
(PeproTech, NJ, USA) (see Note 5).
8. Solution for cell permeabilization and fixation (e.g., BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution, BD
Biosciences, CA, USA).
9. 4
0 ,6-diamidino-2-phenylindole
(DAPI)
dihydrochloride
nucleic acid stain (e.g., Thermo Fisher Scientific, MA, USA).
10. Antibodies for staining MK phenotype markers (e.g., FITCconjugated anti-CD61, BD Biosciences, CA, USA).
2.5 Equipment,
Consumables,
and Reagents for Flow
Cytometry Analysis
1. Flow cytometer (e.g., FACSCanto™ flow cytometry system,
Becton Dickinson, Franklin Lakes, USA).
2. Personal computer with software for acquiring and analysis of
flow cytometry data (e.g., BD FACSDiva™ software, Becton
Dickinson, Franklin Lakes, USA).
3. Sterile polypropylene round-bottom tube for cell separation,
5 mL (e.g., Falcon
® round-bottom polypropylene tubes,
STEMCELL Technologies, Vancouver, Canada).
4. Fluorochrome-conjugated antibodies for immunophenotyping of iPSC-derived MKs (e.g., APC/Cy7-conjugated antiCD41, APC-conjugated anti-CD61, and PE-conjugated antiCD42a, BioLegend, CA, USA).
5. Apoptosis detection kit (e.g., APC Annexin V Apoptosis
Detection Kit with PI, BioLegend, CA, USA).
3 Methods
3.1 Production
and Harvesting
of iPSC-Derived MKs
1. Work in laminar flow hood under sterile conditions.
2. Differentiate iPSCs into MKs by culturing in corresponding
culture medium (e.g., APEL™ Medium) with consecutive
supplementation and change of cytokines and growth factors
according to selected differentiation protocol (see Note 9).
3. On the terminal stage of differentiation, harvest MKs from
differentiation monolayer by gentle rinsing of the monolayer
by careful up and down pipetting and collecting the supernatant with detached cells into 15 mL centrifuge tubes (see Note
10).
4. Centrifuge the supernatant with harvested cells at 120 Â g for
10 min at room temperature in order to concentrate MKs. The
pellet contains the MKs, while the remaining supernatant contains smaller cells and cellular fragments, including the PLTs,
which can be further separated by centrifugation at 750 Â g for
10 min.
542
Denys Pogozhykh et al.
factors for terminal stage of differentiation of iPSCs into MKs
(PeproTech, NJ, USA) (see Note 5).
8. Solution for cell permeabilization and fixation (e.g., BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution, BD
Biosciences, CA, USA).
9. 4
0 ,6-diamidino-2-phenylindole
(DAPI)
dihydrochloride
nucleic acid stain (e.g., Thermo Fisher Scientific, MA, USA).
10. Antibodies for staining MK phenotype markers (e.g., FITCconjugated anti-CD61, BD Biosciences, CA, USA).
2.5 Equipment,
Consumables,
and Reagents for Flow
Cytometry Analysis
1. Flow cytometer (e.g., FACSCanto™ flow cytometry system,
Becton Dickinson, Franklin Lakes, USA).
2. Personal computer with software for acquiring and analysis of
flow cytometry data (e.g., BD FACSDiva™ software, Becton
Dickinson, Franklin Lakes, USA).
3. Sterile polypropylene round-bottom tube for cell separation,
5 mL (e.g., Falcon
® round-bottom polypropylene tubes,
STEMCELL Technologies, Vancouver, Canada).
4. Fluorochrome-conjugated antibodies for immunophenotyping of iPSC-derived MKs (e.g., APC/Cy7-conjugated antiCD41, APC-conjugated anti-CD61, and PE-conjugated antiCD42a, BioLegend, CA, USA).
5. Apoptosis detection kit (e.g., APC Annexin V Apoptosis
Detection Kit with PI, BioLegend, CA, USA).
3 Methods
3.1 Production
and Harvesting
of iPSC-Derived MKs
1. Work in laminar flow hood under sterile conditions.
2. Differentiate iPSCs into MKs by culturing in corresponding
culture medium (e.g., APEL™ Medium) with consecutive
supplementation and change of cytokines and growth factors
according to selected differentiation protocol (see Note 9).
3. On the terminal stage of differentiation, harvest MKs from
differentiation monolayer by gentle rinsing of the monolayer
by careful up and down pipetting and collecting the supernatant with detached cells into 15 mL centrifuge tubes (see Note
10).
4. Centrifuge the supernatant with harvested cells at 120 Â g for
10 min at room temperature in order to concentrate MKs. The
pellet contains the MKs, while the remaining supernatant contains smaller cells and cellular fragments, including the PLTs,
which can be further separated by centrifugation at 750 Â g for
10 min.
542
Denys Pogozhykh et al.