Isolation of Fetal Bone Marrow Cells

The bone marrow of untreated and PNA/DNA NP treated Hbb^th-4/Hbb⁺ mice were collected 3 days post NP delivery on E18.5 as previously reported^{36 (link)}. Freshly isolated fetal bone marrow cells were suspended in ice-cold DMEM + (Dulbecco’s modified Eagle’s medium, 10 mM HEPES, 2% fetal bovine serum [FBS]) at 10⁷ cells ml⁻¹ and stained for 15 min on ice with anti-cKit-PE (eBioscience, Cat #12-1171-82), anti-Sca1-(PerCP)-Cy5.5 (eBioscience, Cat #45-5981-82), and FITC-conjugated lineage marker antibodies [CD4 (eBioscience, Cat #11-0041-82), CD8 (eBioscience, Cat #11-0081-82), Ter119 (eBioscience, Cat #11-5921-82), Gr-1 (eBioscience, Cat #11-5931-82), and CD45 (eBioscience, Cat #11-0452-82)] (Thermo Scientific; Rockford, IL). All antibodies were used at a dilution of 1:100. Samples were then washed with 10 × volume of HBSS + (Hank’s balanced salt solution, 10 mM HEPES, 2% FBS) and centrifuged at 0.4 × g for 8 min at 4 °C. Cell pellets were resuspended in DMEM + and samples were immediately sorted by flow cytometry (BD FACSAria).