Page 328 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
proplatelets; 1:100 dilution: 4.3+2.5% of megakaryocytes made proplatelets) or 12 hr (1:10 dilution: 4.3+1.5% of mega- karyocytes made proplatelets; 1:100 dilution: 5.3+2.5% of megakaryocytes made proplatelets) suggesting PPF does not have an effect from an extracellular environment. Proplatelet
A
initiation was also minimally observed in controls, in which cytoplasm prepared from round non-proplatelet-producing megakaryocytes was cultured with round megakaryocytes at 1 hr (1:10 dilution: 6.0+2.6% of megakaryocytes made proplatelets; 1:100 dilution: 3.2+6.1% of megakaryocytes
BC
DE
Figure 1. Evidence supporting a proplatelet-promoting factor. (A) Overview of approach to identify proplatelet-promoting factor starting with separation of proplatelet-producing megakaryocytes (MK) from round MK. Murine fetal liver cells were recovered from wild-type CD1 mice (Charles River Laboratories) on embryonic day 13.5 and cultured as previously described.11 Fetal liver cell cultures (15,000 MK per fetal liver) were layered on a single-step gradient (1.5-3.0% BSA) on day 4, and MK were allowed to sed- iment for 1 hour (hr). The MK pellet was resuspended in media and cultured for an additional 24 hr, during which proplatelet production was readily observed. MK cultures were layered on a second single-step gradient (1.5-3.0%) on day 5 and intermediate stages in platelet production were resolved within different fractions of the gradient. Proplatelet-producing MK localized to the BSA fraction, whereas round, non-proplatelet-producing MK localized to the pellet. Isolated MK fractions were washed with PBS and then lysed by 3 freeze-thaw cycles and then centrifuged at 100,000 g for 1 hr at 4°C. The supernatant (S100) was recovered and used immediately as the starting material (at 45 mg/mL protein concentration) for microinjection or stored at -80°C until use. A Harvard apparatus PII-100A injector was used for microinjection. Needles were purchased from World Precision Instruments. For each experiment, at least 40 round MK were microinjected with untreated S100 or S100 that was treated as indicated in each experiment. Experiments were repeated in biological triplicates. To determine the percentage of MK making proplatelets, MK were observed on a Nikon Eclipse TS2 microscope with either a 20x/0.40 Ph1 Adl or 40x/0.55 Ph2 Adl objective using an Olympus O-color 3 camera using QCapture Pro 7 software and categorized as round or proplatelet-producing (with cellular extensions) by eye. Data are expressed as percentage of proplatelet-producing MK. Cells were injected with 0.075 nL unless noted otherwise. All data are presented as mean + Standard Deviation (SD). N=3 biological replicates. (B) Representative images showing isolation of round (top) and proplatelet-producing (bottom) MK. Scale bar = 20 μm. (C) Representative images showing microinjection of round MK. Scale bar = 10 μm. (D) Representative images of 1-hr time-point showing microinjection of round MK with cytosol from round MK (left) and cytosol from proplatelet-producing MK (right). Scale bar = 20 μm. (E) Percentage of MK making proplatelets 1 hr post microinjection. N=3 biological replicates. Data are presented as mean + SD. Non-inj: not injected. P<0.0001.
Haematologica | 109 July 2024
2342