Ack hypotonic lysis solution

Bone marrow-derived macrophage cells (BMDMs) were prepared as previously described^{92 (link)}. Briefly, bone marrow cells were flushed from tibiae and femurs of mice. BMDM were differentiated with macrophage colony-stimulating factor (20 ng/mL) containing in DMEM with 10% fetal bovine serum and penicillin/streptomycin over 7 days at 37 °C. To evaluate the role of the ES proteins in macrophage activation, BMDM were left untreated or were treated with 1 μg/mL ES proteins, LPS (100 ng/mL), or IL-4 (20 ng/mL) for 24 h. The cells and culture supernatants were collected and stored at −80 °C for subsequent quantitative real-time PCR. Spleen, mesenteric lymph node (MLN) and lung draining lymph node (LLN) were harvested from mice euthanized by CO₂ inhalation. Peritoneal macrophages were obtained from peritoneal lavage. Cells were isolated as previously reported^{35 (link),36 (link),90 (link),93} . Single cell suspensions were prepared by forcing tissue through a fine wire mesh using a syringe plunger, which was followed by repeated pipetting in RPMI-1640 with 10% fetal bovine serum and penicillin/streptomycin. RBC depletion involved cell lysis in 3 mL ACK hypotonic lysis solution (Sigma-Aldrich).

Following sacrifice, the blood serum of mice was obtained by cardiac puncture and lung-draining lymph nodes (LLNs) and spleen were collected. The LLN in normal mouse (PBS treated mouse) is too small to get enough number of lymphocytes for ELISA experiment, therefore we used only LLN of rAc-PF and Aspergillus protease treated group for ELISA experiment. Each LLN and spleen were ground with MONOJECT and treated with ammonium chloride potassium (ACK) hypotonic lysis solution (Sigma-Aldrich) for 1 min at room temperature for red blood cells lysis. After lysis, lymphocytes and splenocytes were filtered through 100-μm meshes (Small Parts, Inc., Miramar, FL, USA) and then washed three times. Next, the cells were counted with a hemocytometer and plated in 48-well plate as 5 × 10⁶ cells/mL in RPMI 1640 containing 10% fetal bovine serum and penicillin/streptomycin. For CD3 stimulation analysis, 0.5 μg/mL anti-CD3 antibody was added to the plated cells. The plate was incubated at 37°C with 5% CO₂. After 72 h incubation, the culture supernatants were collected and stored at –20°C for enzyme-linked immunosorbent assay (ELISA) [22 (link)].

T cells were isolated from the spleen of normal (control group) or 4T1/TGF-β1 tumor-bearing Balb/c mice with naringenin or 1 % CMCNa treatment. After red blood cells were lysed by ACK hypotonic lysis solution (Sigma), T cells were purified using a pan T-cell isolation kit (Miltenyi Biotech) in accordance with the manufacturer’s protocol. Balb/c nude mice were then intravenously injected with 5 × 10⁵ of these purified T cells mixed with 5 × 10³ 4T1/TGF-β1 tumor cells (100:1 ratio of T cells to 4T1/TGF-β1 tumor cells). T cells with tumor cells were transferred once a week for 3 weeks. The transfer of the mixed cells to nude mice was performed as described previously [30 (link)]. The bioluminescence of lung metastasis in nude mice on day 28 was imaged using the IVIS system as described previously [28 (link)] (details in Additional file 1).