Percp cy5.5 cd45

To quantify various NK cell populations in the PBMCs, 5 × 10⁵ cells were stained with various combinations of fluorophore-conjugated monoclonal antibodies (mAbs). The Gating process of CD3⁻CD16^brCD56^dimNK cells was shown in Figure S1, according to the previous report (36 (link)). Stained cells were fixed with 4% paraformaldehyde and examined by FACSCalibur (BD Biosciences), and the data analyzed Cell Quest Pro software (FlowJo, LLC, Ashland, OR). A total of 50,000 lymphoid events were acquired in each sample. Cells were phenotypically analyzed by staining for 20 min with the following fluorophore-conjugated anti-human mAbs: fluorescein isothiocyanate (FITC)-αCD3, PerCP eFluor710-αCD16, phycoerythrin (PE)-CD56, eFlour660-CD107a, APC-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314(NKG2D), AF647-CD337 (NKp30), and APC-anti-HLA-E (all from BioLegend, eBioscience, and Miltenyi Biotec). Positive staining populations were determined by comparison with isotype controls. To evaluate HLA expression on CD45⁺CD34⁺ myeloid progenitor cells, the cells were stained with PerCP Cy5.5-CD45 (BioLegends), PE-CD34 mAbs (BD Biosciences), and APC-anti-HLA-E mAb, and examined by flow cytometry.

Seventy days after immunization, NOD-EAE mice were anesthetized by intraperitoneal administration of an anesthetic cocktail and transcardially perfused with ice-cold PBS to remove blood. Spinal cords were dissected and minced with a scalpel. Tissues were digested with collagenase D (1 mg/mL, Roche Diagnostics) containing 2.5 mM CaCl₂ at 37 °C for 30 min. Digested tissues were re-suspended in 30% Percoll (GE healthcare). Seventy percent Percoll was overlaid with 30% Percoll, followed by centrifugation at 770 × g for 20 min at room temperature. Immune cells were isolated from the interface of the 30/70% Percoll gradients. The cells were incubated with antibody solution diluted in 2% fetal bovine serum in PBS on ice for 30 min. The following fluorescently labeled antibodies were used: FITC-CD8, PE-CD11b, PerCP/Cy5.5-CD45, APC-Ly6G, and Brilliant violet 421-CD4 (all from BioLegend). Data were collected with a FACSVerse fluidics system (BD Biosciences) and analyzed with FlowJo software (Tree Star).

The experiment was performed using the same procedure as the 22-week heart unless noted here. The antibodies used for staining were PerCP/Cy5.5 CD235a (Biolegend #349110), PerCP/Cy5.5 CD45 (Biolegend #304028), FITC CD36 (Biolegend #336204), APC/Cy7 PECAM1 (Biolegend #303119). The gates were set up to sort cells with low DAPI, low CD45 (hematopoietic cells), low CD235A (erythroid cells), high PECAM1 (endothelial marker), and either low or high FITC. A total of 1824 PECAM1+ CD36-, PECAM1+ CD36+ and PECAM1+ cells from the 11-week heart and 1920 PECAM1+ CD36-, PECAM1+ CD36+ and PECAM1+ cells from the 14-week heart were sorted and processed for cDNA synthesis. A total of 1530 11-week and 1272 14-week cells that had sufficient cDNA concentration were barcoded and pooled for sequencing.
Synthesis of cDNA and library preparation for the fetal human heart cells was performed using the Smart-seq2 method as previously described (Picelli et al., 2014 (link); Su et al., 2018 (link)). Libraries from the fetal human heart cells were part of a pool of samples that was sequenced on four lanes of a Illumina NovaSeq 6000 S4 flow cell.

To assess the BM-MSCs count, cells were isolated from Nck1^+/+ and Nck1^−/− mice as described above and used for flow cytometry analysis at passage 3. BM-MSCs were dissociated using a non-enzymatic dissociation buffer and resuspended in PBS/0.1%BSA. BM-MSCs were stained with the following anti-mouse antibodies: FITC CD31 (Clone: 390; BioLegend 102405), PerCP/Cy5.5 CD45 (Clone: 30-F11; BioLegend 103131), Pacific Blue Sca-1 (clone D7; BioLegend 108119), and PE CD140a (PDGFRα) (Clone: APA5; BioLegend 135905) for 1 h at 4 °C. The stained BM-MSCs were then sorted using a BD FACSCanto II flow cytometer. Data was quantified and analyzed using FACSDiva.

Single‐cell suspensions were prepared from tumor tissues. In brief, the tumor tissues were digested by Hank's buffer containing 200 U·mL⁻¹ DNase type IV, 1 mg·mL⁻¹ collagenase, and 0.1 mg·mL⁻¹ hyaluronidase at 37 °C for 60 min. Single‐cell suspensions were obtained by passing through 40 μm filter (Falcon). Red blood cells (RBC) were removed using RBC lysis buffer (Roche, Penzberg, Upper Bavaria, Germany). Single‐cell suspensions were stained with antibodies accordingly. Antibodies included PerCP‐Cy5.5‐CD45 (BioLegend, San Diego, CA, USA), FITC‐CD3 (eBioscience, San Diego, CA, USA), and PE‐CD8 (eBioscience).