Setup of cocultures of HSPCs with pDNA-loaded CMPs/MkMPs or small RNA–loaded MkMPs

Cocultures of MPs with CD34+ cells were set up as described (27, 28). Briefly, for pDNA delivery, 60,000 HSPCs from day 1 of culture were cocultured with Cy5-pmaxGFP–loaded CMPs or MkMPs with a ratio of 150 CMPs per cell or 20 MkMPs per cell under five different conditions: (i) (Tube) HSPCs and MPs were first cocultured in a small volume (50 to 100 μl) in Eppendorf tubes and diluted in 1-ml coculture medium in 24-well plate (27, 28). (ii) (FT) HSPCs were first seeded onto the upper compartment of the Transwell insert (0.4-μm pore size; Corning). After 30 min, MPs were added into the culture. (iii) (FT + Centri.) HSPC-MP coculture was set up as in (ii) above in the FT system in a 24-well plate. After adding the MPs, the plate was centrifuged under 600g for 30 min at room temperature to physically enhance the contact between HSPCs and MPs. (iv) (FT + PB) HSPCs were pretreated with PB (8 μg/ml) for 15 min at 37°C, followed by coculture with MPs in the FT system as in (ii). (v) (FT + PB + Centri.) HSPCs were pretreated with PB (8 μg/ml) for 15 min at 37°C, followed by coculture with MPs in the FT system with the centrifugation under 600g for 30 min at room temperature, as the combination of (iii) and (iv). The percent of Cy5+ cells at 24 hours and GFP expression at 24, 48, and 72 hours of each coculture were determined by flow cytometry.

For siRNA or miRNA delivery, 60,000 of HSPCs from day 1 of culture were cocultured with MkMPs loaded with siR–Alexa 647, siR-MYB, siR-NC (nontargeting siRNA negative control), miR-486-5p mimic, and miR-NC (nontargeting miR-NC); unloaded MkMPs at a ratio of 30 MkMPs per cell; or without MkMPs as vehicle control in IMDM supplemented with 10% BIT 9500, SCF (50 ng/ml), and 1× antibiotic-antimycotic at 37°C and 20% O2 for up to 8 days. An additional dose of Tpo (10 pg/ml) was added in the siRNA delivery experiment. This minimal amount of Tpo was used to induce c-myb expression so that its down-regulation via siR-MYB can be observed. Cells from the coculture with siR–Alexa 647–loaded MkMPs were harvested at days 1 to 5 for flow cytometric analysis to measure the percent of Alexa 647+ cells. At days 3, 5, and 8, cells cocultured with siR-MYB, siR-NC–loaded MkMPs, or unloaded MkMPs were stained with FITC-conjugated anti-CD41 and APC-conjugated anti-CD34 antibodies for flow cytometric analysis of CD41 and CD34 expression. In miRNA delivery experiment, cells cocultured with MkMPs loaded with miR-486-5p, miR-NC, or unloaded MkMPs were harvested at days 3, 5, and 8 for flow cytometric analysis on CD41 expression. The total cell count and Mk count were calculated at day 8.

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