Anti cd19

Multiplex immunohistochemistry of patient FFPE samples was done in sequential staining cycles using the Opal 7-color Automation IHC Kit (Akoya Biosciences, NEL801001KT) on the BOND RX IHC & ISH Research Platform (Leica Biosystems), which was optimized and performed as described before (33 (link), 34 (link)). The multiplex panel consisted of 1:200 anti-CD14 (Cell Marque, 114R-16) with Opal620, 1:200 anti-CD19 (Abcam, ab134114) with Opal690, 1:150 anti-BDCA2 (Dendritics, DDX0043) with Opal540, 1:100 anti-CD1c (Thermo Fisher Scientific, TA505411) with Opal520, 1:100 XCR1 (Cell Signaling Technologies, 44665S) with Opal570 and 1:1500 anti-pan cytokeratin (Abcam, ab86734) with Opal650. Slides were counterstained with DAPI for 5 minutes and enclosed in Fluoromount-G mounting medium (SouthernBiotech, 0100-01). Whole tissue slides were imaged using the microscope Vectra 3 Automated Quantitative Pathology Imaging System (Version 3.0.4, PerkinElmer Inc.). For comparison to the co-cultures with PDTOs, only DAPI, CD1c, and Pan cytokeratin are shown.

MCF-7 cells were purchased from the Pasture Institute of Iran (Tehran, Iran). Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Co. (Gibco Life Technologies, Carlsbad, CA). Ketamine and xylazine were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The Cobalt-60 facility was provided by the Cancer Institute of Imam Khomeini hospital Complex of Iran (Tehran University of Medical Sciences, Tehran, Iran). Mouse monoclonal antibodies of anti-CD19, anti-CD4, and anti-CD8 were purchased from Abcam CO. INF-γ Mouse ELISA Kit (Abcam; ab46081), IL-4 Mouse ELISA Kit (Abcam; ab100710), and IL-17 Mouse ELISA Kit (Abcam; ab100702) were used for IFN-γ, IL-4, and IL-17 serum level assessment. Total RNA was isolated with a Total RNA extraction kit (GRM1002, VIOGENE, Sunnyvale, CA, USA). Template cDNAs were synthesized using SuperScript III (18080-051, Invitrogen, Carlsbad, CA). Quantitative RT-PCR was performed with a quantitative PCR mix (QPK-201T, Toyobo, Osaka, Honshu, Japan) using a real-time PCR detection system (CFX96; Bio-Rad Laboratories).

Human abdominal subcutaneous adipose tissues were harvested during liposuction procedures after obtaining signed informed consent in the Department of Plastic Surgery, Affiliated Hospital of Zunyi Medical University, China. ADSC isolation and culture was performed as previously described.¹⁵ Briefly, human adipose tissue was minced and digested with 0.075% collagenase type I (Sigma) for 45 minutes at 37°C. After digestion, an equal volume of Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, California) supplemented with 10% fetal bovine serum (FBS; Gibco) was added and the suspension was passed through a 200 μm mesh filter followed by centrifugation at 800g for 5 minutes). The stromal‐vascular fraction cell pellets were resuspended and cultured at 37°C in 5% CO₂ in DMEM supplemented with 10% FBS and 1% penicillin‐streptomycin (Gibco). ADSCs were subcultured at 80% confluence, and passage‐3 cells were used in the study procedures. ADSCs surface marker expression was assayed by flow cytometry. Suspensions of 1 × 10⁶ ADSCs were incubated with anti‐CD90, anti‐CD73, anti‐ CD105, anti‐CD34, anti‐CD11b, anti‐CD19, anti‐CD45, or anti‐HLA‐DR antibodies (1 mg/mL; Abcam) at room temperature for 30 minutes, washed with phosphate‐buffered saline (PBS), and analyzed with a MoFlo XDP flow cytometer (Beckman Coulter, Brea, California) and Kaluza software (Beckman Coulter).