Anti human cd11b percp cy5

BALB/c.Rag2^tmlFwa IL2rg^tmlWjl (Rag2^−/−γc^−/−) mice were bred and maintained in the animal facility of Centro de investigación médica aplicada (CIMA), Universidad de Navarra maintained at CIMA under the guidelines of the Ethics Committee of the center. CHO/mEGFR_mPSMA cells were detached and prestained with CellTracker Green CMFDA Dye (ThermoFisher Scientific; #C7025, 1:1500 dilution) according to manufacturer’s protocol for cells in suspension. The mice were intraperitoneally injected with 1.5 × 10⁶ CMFDA-prestained CHO/mEGFR_mPSMA cells into the left side of the peritoneal cavity. Straight after, they received a separate injection into the right side of the peritoneal cavity containing 3 × 10⁶ human CD11b⁺ cells isolated from a fresh buffy coat as described previously, together with 50 μg of indicated bispecific antibodies. After 24 h, we performed the peritoneal lavage with 3 ml of PBS, and the cell populations were quantified by flow cytometry on CytoFLEX S (BD; B75408). The staining included antimouse CD45.2 (Biolegend #109820, 1:200) in order to exclude murine leukocytes, antihuman CD11b PerCP/Cy5.5 (Biolegend #301328, 1:100) for effector population detection, and Zombie NIR (Biolegend #423105, 1:1000) for live/dead. The gating strategy is shown in Fig. S2.

Alveolar macrophages (M1 or M2) were isolated from human lung samples using the markers mentioned above and described elsewhere. Briefly, after cell dissociation, fluorescence staining was performed using the following antibodies: anti-human CD45-FITC (MHCD4501, 1:100, eBiosciences), anti-human CD206-PE (12-2069-42, 1:100, Invitrogen), anti-human CD1c-APC (17-0015-42, 1:100, Invitrogen), anti-human CD11b-PerCP/Cy5.5 (301327, 1:100, Biolegend), and anti-human CD163-PE-CY7 (25-1639-42, 1:100, Invitrogen). Cells were analyzed in flow cytometers, and data were analyzed using FlowJo Software version 10.8.1 (FlowJo, LLC).