Invivomab anti mouse 4 1bb clone 3h3

The murine melanoma cell line B16-F10 (ATCC) was cultured according to ATCC recommendations and 1 × 10⁵ cells were injected IV in 200 μL RPMI-1640 into C57BL/6 mice to establish a pulmonary metastatic melanoma model. On days 1, 5, and 8 post-inoculation, animals received either 1 × 10⁹ mRBCs IV, 4-1BB agonistic antibody (InVivoMab™ anti-mouse 4-1BB clone 3H3, Bio X Cell, Inc., 2.5 mg/kg) intraperitoneally (IP), or rIL-15 (0.2 mg/kg) IP. On day 14, animals were euthanized and lungs were perfused with ice-cold PBS through the right atrium prior to harvest. The left lobe was collected into RPMI-1640 for immune phenotyping by flow cytometry. Remaining tissue was fixed in 10% buffered formalin for 24 h, switched to 70% ethanol and metastases enumerated under the microscope. For evaluation of frequency of immune cells in the spleen, organs were harvested on day 12 and CD8 + T cells and NK cells were analyzed by flow cytometry.

InVivo MAb anti-mouse 4-1BB (clone: 3H3) and InVivoPlus rat IgG2a isotype control (clone: 2A3) were supplied by BioXCell (New Hampshire, USA). IRDye® 680RD NHS Ester Infrared Dye was supplied by Li-cor (Nebraska, USA). Fixable Viability Stain 780 was purchased from BD Pharmingen (San Diego, USA). Dimethyl sulfoxide (DMSO) Hybri-Max (TM) sterile-filtered BioReagent was purchased from Sigma (St. Louis, MO, USA). The AbC™ total antibody compensation bead kit was acquired from Thermo Fisher (Waltham, MA, USA). PE anti-mouse CD4, FITC anti-mouse CD8a, APC anti-mouse CD137, and Alexa Fluor 594 goat anti-rat IgG (minimal x-reactivity) antibodies were supplied by BioLegend (San Diego, CA, USA). Anti-rabbit IgG (H + L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) was purchased from Cell Signaling Technology (Massachusetts, USA). A purified anti-mouse CD3 antibody was supplied by Thermo (Waltham, MA, USA). An anti-CD137 antibody was purchased from Abcam (Cambridgeshire, UK).