Percp cy5.5 anti cd45

The splenocytes and tumor tissue-derived cells were incubated with Fc Block (#553142, BD Bioscience) prior to staining with PerCP/Cy5.5 anti-CD45 (#103132, Biolegend) and anti-F4/80 antibodies (#45–4801, eBioscience) for 15 min at 4 °C, followed by two washes in Sorting Buffer. Cells were then resuspended in 500 μl of Bodipy 493/503 at 0.5 μg ml⁻¹ in PBS for 15 min. The sorting experiments were performed on FACSAriaII(BD, Bioscience). The Geometric mean fluorescence intensity (MFI) of Bodipy 493/503 in CD45+F4/80+ cells of each group was measured.

SDLN cells were isolated by grinding through a 40 μm strainer to achieve single cell isolation. CD8⁺ T cells were separated from SDLN cells using anti-PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For live/dead staining, the Zombie NIR fixable viability kit (BioLegend, San Diego, CA, USA) was used for 10 min. To block nonspecific binding, cells were incubated with a CD16/CD32 antibody (BD Biosciences, San Jose, CA, USA) for 10 min. For surface staining, the cells were labeled with the following antibodies: PE anti-CD8, anti-CD4; FITC anti-CD8, anti-CD3; APC anti-NKG2D; PerCP-Cy5.5 anti-CD45 (all purchased from BioLegend). The samples were acquired using CytoFLEX (Beckman Coulter, Miami, FL, USA), and the data were analyzed with CytExpert software (version 2.4.0.28).

Tumors were dissociated into single cells and were then filtrated through 70 μm cell strainers. Single cells were resuspended in cell staining buffer (#FXP005, 4abio) and then stained with fluorochrome-conjugated antibody combinations at appropriate concentration. The antibody information are shown as follows: PerCP-Cy5.5-Anti-CD45 (#103131, Biolegend), PE-Cy7-Anti-CD4 (#100421, Biolegend), FITC-Anti-CD8 (#ab237367, Abcam), APC-Anti-TNF-α (#506307, Biolegend), PE-Anti-IFN-γ (#505807, Biolegend), PerCP-Cy5.5-Anti-CD45R (#ab210342, Abcam), and PE-Anti-CD3 (#ab22268, Abcam). DAPI (#D9542, Sigma) was added to exclude dead cells. After washing, the stained cells were analyzed on a BD Fortessa machine. The data were processed with FlowJo software.

To isolate periosteal cells,^{40 (link)} femurs and tibiae were placed in ice-cold PBS after the overlying skin and muscle were carefully removed. The periosteum was gently scratched using a scalpel and forceps and then incubated with prewarmed digestion buffer at 37 °C for 30 min on a shaker. The dissociated periosteal cells were washed twice by centrifugation at 600 × g for 5 min with ice-cold staining buffer.
To isolate growth plate cells,^{28 (link)} dissected growth plates were minced using a scalpel and incubated with 0.15% collagenase (Sigma, C6885) at 37 °C for 90 min on a shaking incubator. The cells were pelleted and resuspended in ice-cold staining buffer.
The cells were stained with PerCP/Cy5.5 anti-CD31 (BioLegend, 102420), PerCP/Cy5.5 anti-CD45 (BioLegend, 103132), PerCP/Cy5.5 anti-mouse Ter-119 (BioLegend, 116228), BV421 anti-mouse CD73 (BioLegend, 127217) or Pacific blue anti-Sca1 (BioLegend, 108120) for 30 min and sorted on an MA900 flow cytometer (Sony) with a 100 μm nozzle.

Bone marrow cells were extracted from femurs and tibias of three WT and six CKO mice at 6-week. Marrow plugs were flushed with a 1-ml syringe using ice-cold DPBS (Corning, 21-031-CMR) containing 2% fetal bovine serum (FBS). These plugs were then dispelled into single cell and the red blood cells were removed by RBC lysis buffer for 5 min (Beyotime, C3702). Wash the cells twice by centrifugation at 600g for 5 min with ice-cold DPBS, 2% FBS. The cells were stained with eFluor 450 anti-CD31 (eBioscience,48-0311-80), PerCP/Cy5.5 anti-CD45 (BioLegend, 103132), APC/Cy7 anti-mouse TER-119 (BioLegend, 116223), FITC anti-mouse 6C3/Ly-51 (BioLegend, 108305), Brilliant Violet 605 anti-mouse CD90.2 (BioLegend, 140317), PE/Cy7 anti-mouse CD105 (BioLegend, 120409), APC anti-mouse CD200 (BioLegend, 123809) for 30 min. Then cells were measured by BC Cytoflex LX after twice of wash by centrifugation at 600g for 5 min with ice-cold DPBS, 2% FBS. The data were then analyzed with the CytExpert software.

Cells were isolated from brain tissue as previously described [20 (link)], with some modifications. Briefly, isolated tissues were cut up into fine pieces and were dissociated in buffer containing HBSS (without calcium/magnesium), 5% FBS, 10 μM HEPES, 2 mg/mL collagenase D (Sigma-Aldrich) and 28U/mL DNaseI (NEB) at 37 °C for 45 min. Every 15 min, dissociated tissue was pipetted up and down using sequentially smaller Pasteur pipettes to homogenize tissue debris. Homogenate was filtered through a 70-μm filter and centrifuged 10 min at 300 g. Pellets were resuspended in PBS and stained with an APC-Cy7 LIVE/DEAD™ fixable dead cell stain kit (Invitrogen). Cells were washed, resuspended in FACS buffer with Fc block (BD Biosciences) and stained with the following antibodies: AF700 anti-CD3 (BioLegend, clone:17A2), BV711 anti-Ly6C (BioLegend, clone: 1A8), BV421 anti-Ly6G (BioLegend, clone: HK1.4), FITC anti-CX3CR1 (BioLegend, clone: SA011F11), BV510 anti-CD19 (BioLegend, clone: 6D5), PerCPCy5.5 anti-CD45 (BioLegend, clone: 30-F11), and APC anti-Cd11b (BioLegend, clone: M1/70). Samples were measured on an LSRFortessa (BD Biosciences) and analyzed using FlowJo software.

Single cell suspensions were washed once with PBS and stained with 100 μL FVD (eBioscience) diluted in PBS for 30 min at 4°C. Cells were washed with equal volume of PBS/2% FCS, followed by Fc blocking with 100 μL anti-human TruStain FcX (BioLegend) in PBS/2% FCS for 10 min at 4°C. Cells were then stained with the PerCP/Cy5.5 anti-CD45 (Biolegend) for 30 min at 4°C and were washed with equal volume of PBS/2% FCS and resuspended in 200 μL PBS/2% FCS for FACS analysis. Data were acquired using an LSR II (BD Biosciences) and analyzed using FlowJo v.10.1 software (TreeStar).