Ack lysis buffer

For flow cytometric analysis and sorting of mouse RANK⁺ MSPCs from femoral metaphysis, we dissected the femora free of soft tissues from mice offspring at 12 weeks old. The epiphysis was removed, and the bones were cut into small pieces and digested in the protease solution for 20 min. Cells within the supernatant were collected for flow cytometry. Cell numbers were determined after removal of red blood cells with ACK Lysis Buffer (CS0001, Leagene, China). Cells were then sorted according to side scatter and RANK-PE fluorescence at >10³ log Fl-1 (RANK-PE) fluorescence. FACS was performed using a 5-laser Beckman FACS system (MoFlo XDF, Beckman, United States). Flow cytometric analyses were performed using FlowJo software (BD Life Sciences, San Jose, CA, United States). The primary antibody used was RANK-PE (12-6612-82, Thermo Fisher Scientific, 5 μL/10⁶ cells).

Peripheral blood mononuclear cells were isolated from whole blood of RAG1, RAG2, and IL2RG mutant piglets and age-matched control piglets. Red blood cells were eliminated by using ACK lysis buffer (Leagene). To identify porcine CD3⁺ T, IgM⁺ B, and CD3^-CD8⁺ NK cells, we used mouse anti-pig CD3 (BD, 561476, 1:200), CD8 (BD, 559584, 1:200), and goat anti-pig IgM (AbD, AAI39F, 1:200). Samples were analyzed using an Accuri C6 flow cytometer (Accuri Cytometers, Ann Arbor, MI).

Bone marrow cells were collected from femurs and tibias of mouse offspring at 4-week-old. Cell numbers were determined after removal of red blood cells with ACK Lysis Buffer (CS0001, Leagene, China). After being washed with PBS twice, pellets were resuspended and blocked in 1% BSA on ice for 15 min. Cells were then washed twice with PBS and incubated with primary antibody (for cell surface marker) solution diluted by 0.5% BSA for 30 min on ice in the dark. After being fixed by 4% paraformaldehyde and permeabilized by PBST and washed with PBS, cells were incubated with primary antibody (for intracellular antigen) solution diluted by 0.5% BSA for 30 min on ice in the dark. The primary antibody used were PE-conjugated Nestin Antibody (MA5-23574, ThermoFish Scientific, Rockford, IL, USA) and BV421-conjugated anti-mouse CD45 (563890, BD Biosciences, San Jose, CA, USA). Cells were then washed once and re-suspended in 300 μl PBS and transferred to flow tubes. For BMSCs, CD45⁻CD29⁺CD105⁺Sca-1⁺ bone marrow cells were detected using a Mouse mesenchymal stem cell Multi-color Flow kit (FMC003, R&D systems, Canada) following the manufacturer’s protocol. Flow cytometric analysis was performed on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD Life Sciences San Jose, CA, USA).

The mice were sacrificed three days after the last treatment. Tumor tissues were collected and cut into pieces. The single cell suspension was prepared by using dissociation buffer (1 mg/mL collagenase IV, 1 mg/ml hyaluronidase, and 20 U/mL DNase in phosphate buffer saline (PBS) plus 2% FBS). The red blood cells were removed by incubating with ACK lysis buffer (CS0001, Leagene). The single cell suspension was stained with Zombie NIR™ Fixable Viability Kit (423105, BioLegend) and immune cell markers (Table S1). Flow cytometry analysis was performed on an LSR Fortessa flow cytometer (BD Biosciences), and data were processed using FlowJo v10 software (TreeStar Inc., USA).

In total, 130 cases of ovarian cancer with malignant ascites were obtained from the Department of Oncologic Gynecology of the Tumor Hospital Affiliated with Harbin Medical University from December 2013 to January 2016. Seventy-six of these patients were not administered chemotherapy, and 54 patients received chemotherapy. All of the cases had complete clinical pathological data: 76.93% (100/130) were the serous type, and the others included mucinous and some other types. Written informed consent was obtained from each patient, and the study was approved by the institutional ethics committee of Harbin Medical University.
ACK Lysis Buffer was purchased from Beijing Leagene Biotechnology Co., Ltd., Beijing, China. The CA125 polyclonal antibody and HE4 polyclonal antibody were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. The CA125 ELISA kit was purchased from Beijing North Institute of Biological Technology Co., Ltd. The HE4 ELISA kit was purchased from Sweden Fujirebio Diagnostics, Inc. The flow cytometry antibody against CA125 was purchased from Biolegend.