The production of IL-34 from HM-1 and 4T1 cell lines was measured by the enzyme-linked immunosorbent assay (ELISA) using LEGEND MAX™ Mouse IL-34 ELISA Kit (Biolegend, San Diego, CA, USA). Cell culture supernatants were collected 48 hours after seeding the cells at a density of 1×106 in a 6 cm dish.
Protein was extracted from HM-1 Mock or Brca1KO tumors and the concentration of IL-34 in each tumor was determined using an ELISA assay. For IL-34 expression analysis in tumors, 2×106 HM-1 cells were inoculated subcutaneously into the flank of syngeneic female mice. Tumors were collected when tumor size reached 10 mm in diameter. From the collected tumors, 10 mg of tumor tissue was weighed using a high-precision analytical balance (AND GH-202) and divided into 500 μL of TNE buffer containing a protease inhibitor. Cell lysates were obtained from the measured tumor tissue which was crushed using a BioMasher® / PowerMasher II (Nippi, Inc., Tokyo, Japan) in TNE buffer. The cell lysate was centrifuged to generate a supernatant sample. Similarly, the concentration of IL-34 in the supernatant sample was determined by ELISA assay.
Protein was extracted from HM-1 Mock or Brca1KO tumors and the concentration of IL-34 in each tumor was determined using an ELISA assay. For IL-34 expression analysis in tumors, 2×106 HM-1 cells were inoculated subcutaneously into the flank of syngeneic female mice. Tumors were collected when tumor size reached 10 mm in diameter. From the collected tumors, 10 mg of tumor tissue was weighed using a high-precision analytical balance (AND GH-202) and divided into 500 μL of TNE buffer containing a protease inhibitor. Cell lysates were obtained from the measured tumor tissue which was crushed using a BioMasher® / PowerMasher II (Nippi, Inc., Tokyo, Japan) in TNE buffer. The cell lysate was centrifuged to generate a supernatant sample. Similarly, the concentration of IL-34 in the supernatant sample was determined by ELISA assay.