Anti cd335

Mice (n = 3) were IP inoculated with 2 × 10⁵ CT-26-Luc cells on Day 4. On Day 0, mice were IP injected with 15 mg/kg luciferin and then imaged using the Lumina IVIS system (PerkinElmer, Waltham, MA, USA) to confirm the presence of tumors in the peritoneal cavity. Mice then received 6 mg/kg OXA via IP injection. The following morning, each mouse received an IP dose of DiI-labeled DSTAP-liposomes at 10 mg lipid/kg. After 1 h, mice were sacrificed, and IP was instilled with 5 mL of ice-cold PBS for PF collection. The PF was centrifuged to obtain IP cells, which were resuspended in a staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide), counted, dispensed into a microtube, and stained with various antibodies (anti-CD45, anti-CD11b, anti-CD11c, anti-F4/80, anti-CD335, anti-CD3, anti-CD8, and anti-EPCAM, BD Life Sciences, Franklin Lakes, NJ, USA) for 30 min on ice in the dark. The cells were centrifuged, resuspended in the fluorescence-activated cell-sorting buffer containing 1 mg/mL propidium iodide, and analyzed by Cytoflex LX (Beckman Coulter, Brea, CA, USA).

Anti-CD4, anti-CD8, anti-CD11c, anti-CD11b, anti-MHC-II, anti-CD80, anti-CD86, anti-CD335, anti-CD3, anti-IFN-γ (all BD Biosciences, Heidelberg, Germany), anti-CD62L, anti-TNF-α, anti-Foxp3 (all eBioscience, Frankfurt, Germany), anti-CD160, anti-CD138, and anti-IL-10 (Biolegend, London, UK) were used as fluorescein isothiocyanate, pacific blue, phycoerythrin (PE), BD Horizon V450, allophycocyanin, AlexaFlour647, PE-cyanin 7, or peridinin-chlorophyll protein conjugates. Dead cells were identified by staining with the fixable viability dye eFlour 780 (eBioscience, Frankfurt, Germany). Intracelluar staining for Foxp3 was performed with the Foxp3 staining kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Cytokine production from freshly isolated splenocytes was measured by stimulating cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, München, Germany) and 100 µg/ml ionomycin (Sigma-Aldrich, München, Germany) for 4 or 6 h (IL-10), respectively, in the presence of 5 µg/ml Brefeldin A (for IFN-γ, TNF-α staining) and 5 µg/ml Monensin (for IL-10 staining), treating with 2% paraformaldehyde and 0.1% NP40, and staining with the respective antibody cocktail. Flow cytometric expression analyses were performed with an LSR II instrument using DIVA software (BD Biosciences, Heidelberg, Germany).