Macsquant x

On day 12, the mice were administered FITC-labeled liposomes or blank liposomes via i.t. injection and sacrificed on day 14. Single-cell suspensions of lung tissue samples were obtained as reported previously (18 (link)). The resuspended cells were stained with stain buffer containing PE-conjugated anti-mouse F4/80 antibodies at 4°C for 30 min. Finally, the samples were washed twice with PBS prior to flow cytometry analysis. Data were acquired on a MACSQuant X (Miltenyi, Bergisch-Gladbach, Germany) and analyzed using FlowJo V10 software.

Mecp2 or Scr siRNA‐transfected BMDMs were digested after IL‐4 stimulation for 24 h and incubated with staining buffer containing APC‐conjugated anti‐mouse F4/80 and FITC‐conjugated anti‐mouse CD206 antibodies at 4°C for 30 min, after which the samples were washed twice with PBS prior to flow cytometric analysis. Mecp2 or Scr siRNA‐treated mice were sacrificed and the lung tissues were collected and digested. After digestion, cells were stained by APC‐conjugated anti‐mouse F4/80, PE‐conjugated anti‐mouse CD11c and FITC‐conjugated anti‐mouse CD206 antibodies. Flow cytometric analysis of liposomes distribution was conducted as follows. On Day 19 after BLM induction, the mice were administered DiO‐labeled liposomes via intratracheal injection and sacrificed on Day 21. The resuspended cells were stained with staining buffer containing APC‐conjugated anti‐mouse F4/80 at 4°C for 30 min. Finally, the samples were washed twice with PBS prior to flow cytometric analysis. Data were acquired on a MACSQuant X (Miltenyi) and analyzed using FlowJo V10 software.

In a 48-well plate 1 × 10⁵ cells were infected with 200 μL Amicon-concentrated lentiviral particles by spinoculation to stably express a gRNA and Cas9-GFP. The next day the cells were split into three wells of a U-bottom 96-well plate and maintained for flow cytometric analysis. The cells were analyzed for GFP expression on MACSQuant^® X (Miltenyi Biotec, Bergisch Gladbach, Germany) every second day starting at 96 h until 14 days after transduction.

Blood cell profiles were assessed using venous blood samples collected at 12–18 months post 0- or 3 Gy whole-body irradiation. The red blood cell distribution width (RDW) was measured using a hematological autoanalyzer XN-1000 (Sysmex, Kobe, Japan). We enumerated the lymphocytes (non-myeloid cells; defined as CD41⁻ CD11b⁻), granulocytes (CD41⁻ CD11b⁺ Ly6C^low), and Ly6C-high monocytes (CD41⁻ CD11b⁺ Ly6C^high) after excluding dead cells stained with 1 μg/mL DAPI (Invitrogen), using a MACSQuant X flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and the FlowJo software (TreeStar, Ashland, OR, USA) as shown in Supplementary Fig. S1. Corresponding antibodies were purchased from BioLegend (San Diego, CA, USA; catalog numbers 101212 for APC CD11b, 133918 for PerCP-cyanine 5.5 CD41, and 128007 for PE Ly6C).

Cytokine concentrations were assessed with commercial kits. Human IFNγ ELISA Set and Human IL-2 ELISA Set (BD, USA) were measured on the Sunrise spectrophotometer and analyzed with Magellan software (Tecan, Switzerland). Interleukin (IL)-2, IL-17a, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), soluble Fas ligand (sFasL), granzyme A, and perforin were measured with the LEGENDplex Human CD8/NK Panel (Biolegend, USA) on the MACSQuant X (Miltenyi Biotec, Germany) and analyzed with Legendplex software (Biolegend, USA).