Facs aria sorp cytometer

Kidneys were harvested and cut into small fragments, and the tissue pieces were collected and dissociated with 1 mg/mL collagenase I (Gibco, USA) for 30 min at 37 °C. Then, the digestion solutions were filtered through 100 μm and 40 μm cell strainers (Falcon, USA) to obtain single-cell suspensions. Primary renal Mφ labeled with F4/80+ were sorted from mice with a FACSAria SORP cytometer (BD Biosciences, USA) and cultured with Mφ medium.
To analyze the phenotype of Mφ, flow cytometric identification was performed using combinations of the following mAbs: F4/80-APC (123116; Biolegend, USA), CD206-PE (141706; Biolegend), and CD11c-FITC (N418; Biolegend). After incubation for 30 min, flow cytometry analysis was performed on a CytoFLEX LX flow cytometer (Beckman Coulter, USA).
To detect the expression of LC3 in kidney-infiltrated Mφ (KM), the cells were blocked with Fc Block (BD Bioscience, USA) for 5 min and stained with the following antibodies: F4/80-PE (QA17A29; Biolegend) and LC3B (E5Q2K; CST). The concentration-matched mouse mAb IgG2b (E7Q5L; CST) was used as the isotype control. Alexa Fluor® 594-conjugated goat anti-mouse IgG (Poly4053; Biolegend) was used as a secondary antibody.

A single-cell suspension of cultured cell lines was obtained by trypsinization (0.25% trypsin, Lonza) at 37°C for 2–3 min. For cell membrane staining cells were resuspended in Hank's balanced salt solution w/o Ca²⁺/Mg²⁺ (HBSS), 2% FBS, 10 mmol/L HEPES pH 7.4 buffer (10⁶cells/100 μL/test) and incubated with LIVE/DEAD® Fixable Dead Cell Stains (Invitrogen, Life Technology Ltd.; 1 μg/mL) and appropriate pre-conjugated antibodies (CD133/1-PE, Miltenyi Biotec, Bergisch Gladback, Germany, 10 μL/test; CD15-PE Immunotools, Friesoythe, Germany, 10 μL/test; A2B5-APC Miltenyi Biotec, 10 μL/test; CD44-FITC, Immunotools, 10 μL/test) 30 min in the dark. Data acquisition was performed with a FACS Aria™ SORP cytometer (BD Biosciences, San Jose, CA) and flow graphs were prepared with the FlowJo software (Tree Star, Inc., Ashland, OR).

HCC cells were detached and suspended at 1 × 10⁶ cells/mL in DMEM medium supplemented with 2% FBS and 10 mM HEPES (pH 7.4). These cells were incubated at 37°C for 90 minutes with 5 μg/mL Hoechst 33342 (Sigma-Aldrich), either alone or in the presence of 5 μM of ABC-transporter inhibitor Reserpine (Sigma-Aldrich). After incubation, the cells were co-stained with 5 μg/mL propidium iodide for dead cell exclusion (BD Pharmingen^™). The cells were rinsed with ice-cold PBS supplemented with 2% FBS and 10 mM HEPES (pH 7.4) buffer. Cell analysis and data acquisition was performed with FACS Aria SORP cytometer (BD Biosciences) using DIVA software.

Animals were perfused with ice-cold PBS. PDOX brains were dissected into separate zones when specified: tumor core (cellular tumor, including pseudopalisading/hypoxic zone if present), invasive zone (corpus callosum and top left hemisphere, P3) and distant zone (left hemisphere, bottom hemisphere, P3 & P13). P8 PDOX was not dissected, due to its very invasive nature. PDOX tumors and control mouse brains were dissociated with MACS Neural Tissue Dissociation Kit (P) (Miltenyi) following the manufacturers’ instructions. Single cells were resuspended in ice-cold HBSS, 2% FBS, 10mM HEPES buffer (100 μl/test) flow buffer. Fc receptors were blocked with CD16/CD32 antibody for 30 min at 4°C. Cells were incubated with the appropriate pre-conjugated antibodies for 30 min at 4°C in the dark (Table S2). Non-viable cells were stained with Hoechst (0.1μg/ml, Sigma). Data acquisition was performed at 4°C on a FACS Aria^™ SORP cytometer (BD Biosciences) fitted with a 632 nm (30 mW) red laser, a 355 (60 mW) UV laser, a 405 nm (50 mW) violet laser and a 488 nm (100 mW). Data were analyzed with FlowJo software (version 10.8.1).

A single-cell suspension was prepared from the nascent tissue of the suture-bony composite defects implanted with PLA for 6 weeks. The cells were incubated with the corresponding primary antibodies (Table S2) at room temperature for 30 min. The supernatant was discarded. The cells were washed twice in BD Perm/Wash™ Buffer, and subsequently incubated in the appropriate second antibodies (Table S2) at room temperature for 10 min. After another 2 rounds of washing, the samples were resuspended in PBS and stored at 4 °C in the dark. A FACSAria SORP cytometer (BD Biosciences, San Jose, CA, USA) was used for sample processing. The proportion of Cd51⁺, Pdgfrα⁺, and Ctsk⁺ cells among Cd45^- cells were analyzed using Flowjo v10.8.1. In vitro-expanded MSC were subjected to the same processing steps to formulate FCM gating strategies (Fig. S6).

6 weeks post-surgery, nascent tissue from the suture-bony composite defects, implanted with GelMA, CTS, and PLA, or without any scaffold (NC), was isolated and subjected to digestion in type 1 collagenase (3.5 mg/mL) at 37 °C for 1 h. Subsequently, tissue debris was removed. The digested cells were collected by centrifugation (500 g, 10 min) and then incubated in Red Cell Lysis Buffer at room temperature for 2 min. Following this, the cells were fixed and permeabilized using BD Cytofix/Cytoperm^TM Fixation/Permeabilization kit at 4 °C for 10 min. After another centrifugation (500 g, 5 min), the cells were incubated with CD45 (clone 30-F11) Brilliant Violet 510 (Table S2) at room temperature for 30 min. The supernatant was discarded. The cells were washed twice in BD Perm/Wash™ Buffer, resuspended in PBS, and stored at 4 °C in the dark. Cd45^- cells were sorted (Fig. 3a) by FACSAria SORP cytometer (BD Biosciences, San Jose, CA, USA) for further transcriptome analysis. The total RNA extraction, RNA-seq, and bioinformatic data analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The accession number for the RNA-seq data reported in this paper is GSE249260.

Data acquisition was performed on a FACS Aria^TM SORP cytometer (BD Biosciences) fitted with a 640 nm (30 mW) red laser, a 355 nm (60 mW) UV laser, a 405 nm (50 mW) violet laser, a 488 nm (100 mW) blue laser, and a 561 nm (50 mW) yellow/green laser. The Hoechst dye was excited by the UV laser and fluorescence was collected in two channels: UV-1 450/50 band-pass (BP) filter and UV-2′ 660/40 long-pass (LP) filter. An LP 635 nm dichroic mirror was used to split the emission wavelengths. The instrument was calibrated each time with Cytometer Setup&Tracking Beads (BD Bioscience). The Coefficient of Variation of the instrument (% CV) was routinely examined before each experiment. A 100 µm nozzle and window extension (WE) 3 were used for data acquisition and sorting. Imaging flow cytometry was performed with an ImageStream imaging cytometer (Amnis) fitted with a 375 UV laser, a 488 blue laser, a 561 yellow-green laser, a 642 red laser, and a 785 nm infrared laser. Acquisition was performed with the INSPIRE^® software and analysis was performed using IDEAS^® image analysis software. Pictures were taken at ×60 magnification at low-speed high-sensitivity mode.