Cell strainer 70 m nylon

Tumor inoculation and the treatment were performed as described earlier. Sera were serially collected from the mice before, and at 6 and 24 hours after PD-L1-targeted NIR-PIT on MC38-luc tumors. Tumors were harvested and homogenized in 1 mL PBS supplemented with protease inhibitors (cOmplete Tablets; Sigma-Aldrich, St. Louis, Missouri, USA); then, the solution was passed through a filter (Cell Strainer 70 µm Nylon; Corning, Corning, New York, USA). Concentrations of various cytokines and chemokines in the samples were analyzed with Mouse Cytokine Array/Chemokine Array from Eve Technologies (Calgary, Canada).

Tumors and spleens were removed with scissors or forceps and weighted. Tumors were chopped into fine pieces and transferred into RPMI media (5% FBS + 10 mM HEPES) containing collagenase (0.2 mg/mL) (Clostridium histolyticum, Sigma) and spleens were suspended in PBS. Tumors were shaken gently at 37 °C for 30 min prior to tissue disruption. Spleen and tumor tissues were disrupted using a cell strainer (70 µm nylon) (Corning) using a syringe plunge. Nylon mesh was rinsed several times with media. Samples were spin (5 min at 1500 rpm) and re-suspended in 1 mL RBC lysis buffer (Sigma) for 5 min and washed 2× with flow buffer (Thermo Fisher). Cells were counted (Nexelom Cellometer), prior to live/dead staining with Zombie UV (Biolegend), incubated for 15 min at room temperature and washed 2× with PBS. This was followed by Fc block (CD16/CD32, BD Biosciences), antibody staining and preparation of single-color compensation controls (Ultra Comp eBeads compensation beads, Thermo Fisher). Samples were covered in foil and kept at 4 °C overnight. Intracellular staining was performed using Permeabilization Buffer (eBioscience) in conjunction with Foxp3/Transcription Factor Staining Buffer set (eBioscience). Fluorescence minus one (FMO) controls were used where indicated to distinguish between positively and negatively stained cells for FoxP3, Ly6G, and Ly6C.