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Alexafluor700 f4 80

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AlexaFluor700-F4/80 is a fluorescently labeled antibody reagent used in flow cytometry applications. It recognizes the F4/80 antigen, which is a cell surface glycoprotein expressed on macrophages and related cell types. The AlexaFluor700 fluorophore allows for detection in the near-infrared range.

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F4/80 (201 citations)

2 protocols using alexafluor700 f4 80

1

Monocyte-derived Macrophage Phenotyping

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Bone marrow cells were flushed from femurs and tibias, centrifuged, then exposed to geys buffer for 5 min on ice, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with antibodies for 20 min at 4 °C, and washed before sorting CD115+/CD11c-/NK- monocytes using an Influx cell sorter (BD) [68 (link)]. Monocytes were cultured in complete RPMI medium (Thermo Fisher Scientific) and exposed to 100 ng/mL CSF1, 50 µM Q-VD-OPh and 50 µM Ac-YVAD-cmk. After five days, macrophages were studied by flow cytometry using AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BUV737-CD43 (#564398, BD). For the sorting of monocytes, the antibodies used were the following: APC-CD11b (#17-0112-83, Thermo Fisher Scientific), PE/Cy7-CD11c (#561022, BD), FITC-NK (#553164, BD), PerCP/Cy5.5-Ly6C (#560525, BD), AlexaFluor700-Ly6G (#561236, BD), biotin-CD115 (#135507, Biolegend), PE-Streptavidin (#554061, BD). The data were analyzed with FlowJo software v. 10.0.00003.
Solier S., Mondini M., Meziani L., Jacquel A., Lacout C., Berghe T.V., Julé Y., Martinou J.C., Pierron G., Rivière J., Deloger M., Dupuy C., Slama-Schwok A., Droin N., Vandenabeele P., Auberger P., Deutsch E., El-Benna J., Dang P.M, & Solary E. (2023). Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues. International Journal of Molecular Sciences, 24(4), 4151.
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2

Interstitial Macrophages Identification

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Right lungs were digested with the Lung Dissociation kit (Miltenyi Biotec), filtered, erythrocytes were removed using ACK and nucleated cells were collected. Cells were washed with ice-cold PBS, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with the antibodies for 20 min at 4 °C, washed, then fluorescence was measured with a BD LSRFortessa X-20. Antibodies used were the following: AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), FITC-CD45 (#103108, Biolegend), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BV650-CD24 (#563545, BD), BUV737-CD43 (#564398, BD). The data were analyzed with FlowJo software v. 10.0.00003. Interstitial macrophages were selected according to their larger size (FSC) and granularity (SSC), on CD45 expression to select leukocytes and GR1-positive cells will be excluded to remove neutrophils, they are equally CD11b high, SiglecF negative, IA-IE positive and CD24 negative.
Solier S., Mondini M., Meziani L., Jacquel A., Lacout C., Berghe T.V., Julé Y., Martinou J.C., Pierron G., Rivière J., Deloger M., Dupuy C., Slama-Schwok A., Droin N., Vandenabeele P., Auberger P., Deutsch E., El-Benna J., Dang P.M, & Solary E. (2023). Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues. International Journal of Molecular Sciences, 24(4), 4151.
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