Invivomab anti mouse pd 1

PRT, OVA, propidium iodide (PI) and lipopolysaccharides (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CpG 1826 (5′-TCC ATG ACG TTC CTG ACG TT-3′) was bought from Sangon (Shanghai, China). Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) from mouse were bought from Novus Biologicals (Colorado, USA). The antibodies such as CD11c-PE, CD86-APC, CD80-FITC, CD3e-PE, CD8a-FITC and OVA_257-264 (SIINFEKL) peptide bound to H-2 K^b APC for flow cytometry (FCM) detection, and the biotinylated OVA_257-264 (SIINFEKL) peptide bound to H-2 K^b monoclonal antibody for immunofluorescence, these antibodies were all bought from eBioscience (CA, USA). The ELISA kits for mouse interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were bought from R&D Systems (Minneapolis, MA, USA). The aPD-1 (InVivoMab anti-mouse PD-1) was purchased from BioXcell (New Hampshire, USA). Avidin-FITC was bought from Boster Bio-Tech (Wuhan, China).

Female BALB/c mice (8-week-old) were inoculated with 1 × 10⁶ 4T1-Luc cells in 100 μl of PBS by subcutaneous injection into the upper right mammary fat pad. Tumors were measured with calipers, and tumor volume was calculated according to the formula (1/2 × length × width²). The tumor-bearing mice were randomly allocated to the control and treatment groups on day 6 after implantation. For the cell treatment groups, the mice received intravenous injected 1 × 10⁶ engineered macrophages in 100 μl of PBS per mouse per injection every 3 days for 12 days. The day after cell injection, the mice in antibody treatment groups received intraperitoneal injected 50 μg of aPD-1 (InVivoMab anti-mouse PD-1, Bio X Cell) per mouse per injection. Engineering BMDMs were acquired from BALB/c mice as previously described, and M1-polarized macrophages were engineered by incubating with ARMFUL for 4 hours in serum-free DMEM. To monitor tumor growth, the mice received in vivo imaging on days 2, 7, and 12 after the first injection. Briefly, mice were administrated with 200 μl of d-luciferin potassium salt bioluminescence substrate (15 mg/ml; Yeasen) in PBS and 200 μl of pentobarbital sodium (0.6 wt %) in PBS via intraperitoneal injection. Ten minutes after injection, mice were imaged under anesthesia using an in vivo imaging system (AniView600, BLT Photon Technology).