Cm dii

To assess endogenous levels of osteoblastic mRNAs in osteogenically differentiating MSCs in contact with OBCs, MSCs and OBCs were co-cultured in a 1:4 ratio at 9,000-10,000 cells/cm². Before co-culture, trypsinized MSCs were stained with the cell tracker CM-DiI (Thermo Fisher, Waltham, MA; 10⁶ cells/mL + 8 μL CM-DiI, 37 °C, for five min, then 4 °C, for 15 min) to distinguish them from OBCs and to allow subsequent fluorescence-activated cell sorting (FACS). Three co-culture combinations were tested in both EM and OM: (1) DiI-labeled MSCs mixed with unlabeled OBCs, (2) DiI-labeled MSCs mixed with unlabeled MSCs, and (3) unlabeled, MSC-only cell populations, with the latter two conditions serving as controls. Samples were harvested pre-culture, and at culture days 6 and 12. MSC-only cultures were trypsinized, centrifuged, resuspended in 1 mL Trizol (Invitrogen), and stored at -80 °C; mixed cultures were trypsinized and frozen in dimethyl sulfoxide (DMSO) freeze medium (BioVeris Corporation, Gaithersburg, MD, USA) until the time course was complete. The DiI-positive cells were fractionated using a Becton-Dickinson three-laser Dako MoFlo FACS sorter, pelleted by centrifugation, resuspended in 1 mL Trizol, and stored at -80 °C. Osteogenesis was assessed by conventional real time RT-PCR and by PCR array analysis (SuperArray, Qiagen, Valencia, CA, USA; see below).

All animal work complied with protocols approved by the Institutional Animal Care and Use Committee at the Marine Biological Laboratory (MBL; Woods Hole, MA, USA). Skate (Leucoraja erinacea) eggs were obtained from the Marine Resources Centre of the MBL. Staging was carried out according to Maxwell et al. (2008) (link) and Ballard et al. (1993) (link). Embryos for histological and gene expression analyses were fixed in 4% paraformaldehyde (PFA) in 1× phosphate-buffered saline (PBS) overnight at 4°C, then rinsed in 1× PBS, dehydrated into methanol and stored in methanol at −20°C prior to analysis. Embryos for cell-lineage tracing experiments were maintained in a flow-through seawater system at ∼15°C. For CM-DiI (ThermoFisher) labelling of the mandibular arch at S22, eggs were windowed and microinjection of the mandibular arch with CM-DiI was performed in ovo according to Gillis and Tidswell (2017) (link). For labelling of the mandibular arch at S27, embryos were removed from the egg and anaesthetised in a petri dish of seawater containing 10 mg/ml ethyl 3-aminobenzoate methanesulfonate salt (MS-222, Sigma) prior to microinjection, according to Gillis and Hall (2016) (link). CM-DiI-injected embryos were left to develop for 6-10 weeks, and then fixed in 4% PFA in PBS overnight at 4°C, rinsed three times in PBS and stored at 4°C in PBS with 0.01% sodium azide before analysis.

The recombinant lentiviruses (Lenti) carrying murine CCR5 (Lenti-EGFP-CCR5) or a control transgenic EGFP (Lenti-EGFP) were prepared as previously described [19 (link)]. Passage 2 of EPCs was used for lentivirus transfection. Prior to transfection, cultured EPCs were labeled with CM-DiI (4 mg/mL; Molecular Probes, Eugene, OR, USA) for 15 minutes in accordance with the protocol of the manufacturer. Then the EPCs were incubated in EBM-2 containing Lenti-EGFP-CCR5 or Lenti-EGFP particles. The lentiviral particles were removed after 24 hours. The double-labeled EPCs were harvested (passage 3) and resuspended at 1 × 10⁶ cells/mL in PBS for administration to the mice.

Human monocyte cell line THP‐1 was cultured in RPMI 1640 supplemented with 10% FBS (Gibco) and cultured at 37°C in humidified 5% CO₂ incubator. The THP‐1 cells were pre‐labelled with CM‐DiI (2 μM, Molecular Probes, Thermo Fisher Scientific) for 5 min. at 37°C and then for an additional 15 min. at 4°C in RPMI 1640. After treatment, the THP‐1 cells were added to each well of HUVECs and co‐cultured for 2 hrs. Each well was washed three times with PBS, and then, images were randomly captured at ten regions in each well at 200 × magnification using fluorescence microscope (IX71, Olympus, Japan).

BMDMs (2 × 10⁶) from WT or p85α+/− mice were intraperitoneally injected into WT mice 2 days before and 1 day after the start of DSS treatment. To neutralize IL-10, LEAF Purified anti-mouse IL-10 or LEAF Purified Rat IgG1κ (250 μg/mouse; Biolegend, San Diego, CA) was injected intraperitoneally into p85α+/− BMDM-transferred WT mice 2 h before the start of DSS treatment and then every other day. To trace the transferred BMDMs in vivo, BMDMs were labeled with the fluorescent membrane marker CM-DiI (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions and injected into mice. After 2 days, cLP cells were isolated from the mouse colons and the proportion of the CD11b⁺F4/80⁺CM-DiI⁺ intestinal macrophages was analyzed using a FACSCanto II flow cytometer.