Phycoerythrin conjugated anti f4 80

Cells were collected from BALF and stained with the following antibodies for flow cytometry: fluorescein isothiocyanate-conjugated anti-CD11b, allophycocyanin-conjugated anti-CD11c, phycoerythrin:Cy-7 conjugated anti-Ly 6G, and phycoerythrin-conjugated anti-F4/80 (BD Biosciences). Cells were sorted on a FACSVerse instruments (BD Biosciences) and data were analyzed with BD FACSuite software.

Total leukocyte counts in BALF were determined using a hemocytometer. Myeloid cells were quantified by incubating BALF cells with purified anti-mouse CD16/32 (BD Biosciences, USA) and stained with allophycocyanin (APC)-conjugated anti-CD11b (BD), FITC-conjugated anti-Ly-6G (BD), FITC-conjugated anti-CD11c (BioLegend, USA) and phycoerythrin-conjugated anti-F4/80 (BD).
To differentiate early-stage apoptotic cells from late-stage apoptotic and necrotic cells, BALF cells were stained with both FITC Annexin V and PI (BD Biosciences) in accordance with the manufacturer’s instructions. To evaluate necrosis induction in macrophages, cells were stained using PI, 7-AAD (BD), and macrophage-specific antibodies (see above). Subsequently, alveolar macrophages were gated according to their FSC/SSC, F4/80-PE, and CD11c-FITC cell surface expression, and other macrophages were gated according to their FSC/SSC, F4/80-PE, and CD11b-APC cell surface expression, followed by determination of the percentage of PI/7-AAD⁺-resistant alveolar macrophages and recruitment macrophages as previously described (34 (link)).

Total leukocyte counts in BALF were determined using a hemocytometer. Myeloid cells were quantified by incubating BALF cells with purified anti-mouse CD16/32 (BD Biosciences, USA) and stained with allophycocyanin (APC)-conjugated anti-CD11b (BD), FITC-conjugated anti-Ly-6G (BD), FITC-conjugated anti-CD11c (BioLegend, USA) and phycoerythrin-conjugated anti-F4/80 (BD).
To differentiate early-stage apoptotic cells from late-stage apoptotic and necrotic cells, BALF cells were stained with both FITC Annexin V and PI (BD Biosciences) in accordance with the manufacturer’s instructions. To evaluate necrosis induction in macrophages, cells were stained using PI, 7-AAD (BD), and macrophage-specific antibodies (see above). Subsequently, alveolar macrophages were gated according to their FSC/SSC, F4/80-PE, and CD11c-FITC cell surface expression, and other macrophages were gated according to their FSC/SSC, F4/80-PE, and CD11b-APC cell surface expression, followed by determination of the percentage of PI/7-AAD⁺-resistant alveolar macrophages and recruitment macrophages as previously described (34 (link)).