Single cells were directly sorted into 96-well plates with erythroid/megakaryocyte bipotential medium for erythroid and megakaryocytic output (Psaila et al., 2016 (link)). StemSpan (StemCell Technologies, Inc.) was supplemented with thrombopoietin (TPO; 100 ng/ml), erythropoietin (EPO; 1 IU/ml), stem cell factor (SCF; 100 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml; all from Peprotech), and human low-density lipoprotein (40 mg/ml; StemCell Technologies, Inc.). Single cells were cultured at 37°C in 5% CO2 for 2 wk, and colony readout was performed by direct light microscopy and flow cytometry using CD41a APC (eBioscience; Clone: HIP8, Cat# 17–0419-42, RRID: AB_2573144), CD42b PE (eBioscience; Clone: HIP1, Cat# 12–0429042, RRID: AB_10852864), CD11b FITC (eBioscience; Clone: ICRF44, Cat# 11–0118-42, RRID: AB_1582242), CD14 FITC (eBioscience; Clone: 61D3, Cat# 11–0149-41, RRID: AB_10597445), CD34 APC–eFluor 780 (Clone: 4H11, Cat# 47–0349-42, RRID: AB_2573956), and CD235a PerCp Cy5.5 (Biolegend; Clone: HIR2, Cat# 306614, RRID: AB_10683170). CD41a and CD42b double-positive cells were used to identify the megakaryocytic output.
Protocol detail
Single-cell bipotent erythroid-megakaryocyte culture
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Cd11b
Cd235a
Cells
Clone
Cy5 5
Erythropoietin
Fitc
Flow cytometry
Hip1
Human
Light microscopy
Low density lipoprotein
Megakaryocytic
Stem cell factor
Stemcell
Thrombopoietin
Corresponding Organization : Churchill Hospital
Other organizations : Imperial College London, St Mary's Hospital, Great Ormond Street Hospital for Children NHS Foundation Trust, National Health Service, MRC Human Immunology Unit
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Variable analysis
independent variables
- Single cells were directly sorted into 96-well plates with erythroid/megakaryocyte bipotential medium
- Erythroid and megakaryocytic output
- CD41a and CD42b double-positive cells (megakaryocytic output)
- Cells were cultured at 37°C in 5% CO2 for 2 weeks
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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