Megacult c kit

Single-cell assays testing for My, Ery, Meg and Ly differentiation were performed as described in ³⁵ . Single-cell cultures specifically promoting Ery and Meg lineage differentiation were modified from a published protocol ^{36 (link)}. Bulk colony assays were performed using the Megacult^™-C kit or MethoCult^™ (H4034) medium following manufacturer’s instructions (Stem Cell Technologies). Further details on cell culture assays and their analysis are described in the supplemental methods.

Colony assays for quantifying human megakaryocytic progenitors were performed using the MegaCult‐C kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer's instructions. Human CD34⁺ cells were suspended in a double‐chamber slide (5.0 × 10³ cells/well), with hetrombopag, eltrombopag or rhTPO. After incubation for 10 days at 37°C in a humidified chamber with 5% CO₂, CFU‐MK was detected by staining the cells with anti‐human CD41 antibody. The number of CFU‐MK was counted and subdivided by colony size, and classified as small (3‐20 cells/colony), medium (21‐49 cells/colony) and large (50 cells/colony).

Bone marrow and splenic mononucleated cells were incubated in duplicate at cell concentrations of 2 × 10⁴ and 2 × 10⁵/mL, respectively, in MethoCult-M3434 semisolid culture medium (STEMCELL TECHNOLOGIES). Colonies were scored on day 3 for erythroid colony-forming units (CFU-Es) and on day 8 for granulocyte/erythroid/monocyte/megakaryocyte colony-forming units (CFU-GEMMs), granulocyte/monocyte colony-forming units (CFU-GMs), and monocyte colony-forming units (CFU-Ms). For the megakaryocyte colony-forming units (CFU-Megs) assays, 5 × 10⁴/mL bone marrow and 5 × 10⁵/mL spleen cells were cultured with MegaCult-C Kit (STEMCELL TECHNOLOGIES) for 9 days, and colonies were stained for acetylcholine esterase (AChE) in accordance with manufacturer’s recommendation.