Cd45 pe

For MSC cell-surface characterization, the following antibodies were used: APC-Vio770 CD29 (Miltenyi Biotec; clone, HMβ1-1), VioBright FITC CD44 (Miltenyi Biotec, USA; clone, REA665), PerCP Sca-1 (Biolegend, USA; clone, D7), PE CD31 (Miltenyi Biotec, USA; clone, 390), PE CD45 (Biolegend, USA; clone, 30-F11), and PE-Vio770 CD105 (Miltenyi Biotec, USA; clone, MJ7/19). For analysis of in vitro macrophages, we first blocked Fc receptors with CD16/32 FcγRIII/FcγRII (BD Bioscience, USA; clone, 2.4G2) and CD16.2 FcγRIV (Biolegend, USA; clone, 9E9) and then stained with PE CD45 (Biolegend, USA; clone, 30-F11), Alexa Fluor 647 CD206 (BioLegend, USA; clone, C068C2), and PE CD163 (ThermoFisher, USA; clone, TNKUPJ). A Novocyte 2060 (ACEA Biosciences, USA) was used to measure mean fluorescence intensity of GFP from transfected MSCs, MSC cell-surface markers, and macrophage markers. Data was analyzed in FlowJo software (BD Biosciences, USA).

To detect cell surface Gal-9 expression, the cells were resuspended in phosphate-buffered saline (PBS) and stained with APC anti-mouse Gal-9 antibody (BioLegend) as previously described using standard protocols for flow cytometry 17 (link). An isotype IgG antibody was used as a negative control. Stained cells were evaluated using a BD FACSCanto II cytometer, and data were analyzed using FlowJo software. To analyze cytotoxic T lymphocyte (CTL) profiles and Gal-9 levels in mouse tumor samples, a Mouse Tumor Dissociation Kit (Miltenyi Biotec) and gentleMACS Octo Dissociator (Miltenyi Biotec) were used to digest mouse tumors into single cells. After the removal of red blood cells and hybridization with CD16/CD32 antibody (TruStain fcX, BioLegend), single cells were stained for flow cytometry according to standard protocols with antibodies against the following: PE-CD45 (BioLegend), PerCP-CD3 (BioLegend), APC/Cy7-CD8a (BioLegend), and APC-Gal-9 (BioLegend). For further intracellular staining, cells were fixed, permeabilized, and stained with Pacific Blue granzyme B (BioLegend). Stained cells were analyzed using a BD FACSCanto II cytometer (BD Biosciences). Data were analyzed by using the FlowJo software program.

The captured, alive CTC clusters were stained with Alexa 488-conjugated antibodies against EpCAM (Cell Signaling Technology – Cat No: 5198S, (1:400)), prostate-specific membrane antigen (PSMA) (Biolegend – Cat No: 342506, Clone: LNI-17, (1:80)), and the contaminating WBCs were stained with PE-CD45 (TRITC) (BioLegend – Cat No: 368510, Clone: 2D1, (1:40)). The device with captured clusters was mounted in a Petri dish, and then imaged using inverted fluorescence microscope (Eclipse Ti, Nikon, Melville, NY). Identified clusters were directly micromanipulated from the device and transferred to the PCR tubes containing RLT (Qiagen) + BME (Sigma-Aldrich) buffer using Eppendorf TransferMan 4r micromanipulator. During micromanipulation, the dish was supplemented with 1× PBS to prevent microwells from drying, which was observed to take ~10 min at room temperature, Tubes were vortexed for 1 min and transferred to −80 °C freezer. Most of contaminating WBCs were observed to remain adhered to the device during cluster retrieval. Nevertheless, CTC clusters micromanipulated from Cluster-Wells were discharged into an empty Petri dish and were then repicked to ensure against potential artifacts from WBC contamination in RNA sequencing.