Functions of BMF-derived MKs. (A) Representative electron microscopy images of enriched MKs from normal mice and BMF mice. N, nucleus (black arrows); DMS, demarcation membrane system (green arrows); proplatelets (P, red arrows); α-granules (A, purple arrows). Scale bars = 2 μm under 1200× magnification, and 2 μm under 3000× magnification, respectively (from pooled 15 BMF mice and 10 normal mice, respectively). (B) Comparison of platelet production between BMF MK and normal control MKs. MKs (3 × 104/mL) from pooled 10 BMF mice and 5 normal mice were seeded into 96-well plates, respectively, and cultured for 6 days as described previously,19 then counted manually. (C) BMF-derived MKs suppress colony forming capacity of normal BM cells. MKs were isolated from pooled samples of BMF mice (n = 30) and control CByB6F1 mice (n = 15). BM cells (2 × 104) from normal CByB6F1 mice were incubated with BMF-derived or normal control MKs (8000 cells) at 37°C for 1 hour, then were mixed in semisolid methylcellulose medium, and plated on 35-mm culture dishes. Cells were cultured at 37°C with 5% CO2. Colonies were counted on day 7. Data shown were from 2 separate experiments. CFU, colony forming unit. (D) BMF-derived MKs induce apoptosis and death of normal BM cells. MKs (2 × 104 cells) isolated from BMF mice (n = 3 pools) or control CByB6F1 mice (n = 2 pools) were incubated with BM cells (2 × 105 cells) from normal CByB6F1 mice at 37°C for overnight, 7AAD and annexin V positivity on BM cells was evaluated by flow cytometry. BMF-MK, normal BM cells cocultured with BMF-derived MKs; NC-MK, normal BM cells cocultured with normal mice-derived MKs. (E) BMF-derived T cells induce upregulation of IA-IE on normal MKs after coculture. Representative plot of IA-IE expression on MKs after coculture with T cells for overnight. Individual value plots of IA-IE on MKs after coculture with T cells. (F) OT-1 CD8+ T-cell response mediated by OVA-1 peptide-pulsed MKs. (G) OT-2 CD4+ T-cell response mediated by OVA-2 peptide-pulsed MKs. Ten thousand flow-sorted normal MKs (a fraction of pooled from 10 mice) or BMF MKs (a fraction of pooled from 15 mice) were pulsed with OVA-1 or OVA-2 peptides (200 μg) for 12 hours. After wash with phosphate-buffered saline, they were cocultured with 1 × 105 LN cells collected from OT-1 or OT-2 mice for an additional 12 hours. OT-1/2 LN cells + BMF MKs or normal control MKs without peptides served as negative controls. MK:LN ratio = 1 × 104:1 × 105. To test MHC-2–dependent OT-2 CD4+ T-cell response, anti-IA-IE antibody (250 μg/mL) was added to MKs before OVA-2 peptide pulsing. Representative flow cytometry plots and results are shown. CD8+ T cells and CD4+ T cells were gated in panels F and G, respectively. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. 7AAD, 7-aminoactinomycin; DMS, demarcation membrane system; NC, normal control; PLT, platelet; SSC-A, side scatter area.

Functions of BMF-derived MKs. (A) Representative electron microscopy images of enriched MKs from normal mice and BMF mice. N, nucleus (black arrows); DMS, demarcation membrane system (green arrows); proplatelets (P, red arrows); α-granules (A, purple arrows). Scale bars = 2 μm under 1200× magnification, and 2 μm under 3000× magnification, respectively (from pooled 15 BMF mice and 10 normal mice, respectively). (B) Comparison of platelet production between BMF MK and normal control MKs. MKs (3 × 104/mL) from pooled 10 BMF mice and 5 normal mice were seeded into 96-well plates, respectively, and cultured for 6 days as described previously,19 then counted manually. (C) BMF-derived MKs suppress colony forming capacity of normal BM cells. MKs were isolated from pooled samples of BMF mice (n = 30) and control CByB6F1 mice (n = 15). BM cells (2 × 104) from normal CByB6F1 mice were incubated with BMF-derived or normal control MKs (8000 cells) at 37°C for 1 hour, then were mixed in semisolid methylcellulose medium, and plated on 35-mm culture dishes. Cells were cultured at 37°C with 5% CO2. Colonies were counted on day 7. Data shown were from 2 separate experiments. CFU, colony forming unit. (D) BMF-derived MKs induce apoptosis and death of normal BM cells. MKs (2 × 104 cells) isolated from BMF mice (n = 3 pools) or control CByB6F1 mice (n = 2 pools) were incubated with BM cells (2 × 105 cells) from normal CByB6F1 mice at 37°C for overnight, 7AAD and annexin V positivity on BM cells was evaluated by flow cytometry. BMF-MK, normal BM cells cocultured with BMF-derived MKs; NC-MK, normal BM cells cocultured with normal mice-derived MKs. (E) BMF-derived T cells induce upregulation of IA-IE on normal MKs after coculture. Representative plot of IA-IE expression on MKs after coculture with T cells for overnight. Individual value plots of IA-IE on MKs after coculture with T cells. (F) OT-1 CD8+ T-cell response mediated by OVA-1 peptide-pulsed MKs. (G) OT-2 CD4+ T-cell response mediated by OVA-2 peptide-pulsed MKs. Ten thousand flow-sorted normal MKs (a fraction of pooled from 10 mice) or BMF MKs (a fraction of pooled from 15 mice) were pulsed with OVA-1 or OVA-2 peptides (200 μg) for 12 hours. After wash with phosphate-buffered saline, they were cocultured with 1 × 105 LN cells collected from OT-1 or OT-2 mice for an additional 12 hours. OT-1/2 LN cells + BMF MKs or normal control MKs without peptides served as negative controls. MK:LN ratio = 1 × 104:1 × 105. To test MHC-2–dependent OT-2 CD4+ T-cell response, anti-IA-IE antibody (250 μg/mL) was added to MKs before OVA-2 peptide pulsing. Representative flow cytometry plots and results are shown. CD8+ T cells and CD4+ T cells were gated in panels F and G, respectively. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. 7AAD, 7-aminoactinomycin; DMS, demarcation membrane system; NC, normal control; PLT, platelet; SSC-A, side scatter area.

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