Purified anti-mouse TNF-α Antibody

Protocol tips

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	BMDMs were differentiated for 24 hours in M0, M1 or M2 conditions and blocked with anti-mouse FcR antibody (CD16/CD32, BD) for 15 min at 4°C in FACS buffer (PBS with 2% FBS and 1 mM EDTA), subsequently cells were stained for 15 min at 4°C with blue-fluorescent reactive dye. Cells were washed three times with FACS buffer, fixed with the eBioscience Fixation/Permeabilization buffer for 40 min at 4°C and washed three times in 1X eBioscience Permeabilization buffer. For intracellular staining, cells were first blocked with anti-mouse FcR antibody (CD16/CD32, BD) in 1X Permeabilization buffer (eBioscience) for 45 min at 4°C prior to staining.

Protocol tips

BMDMs were differentiated for 24 hours in M0, M1 or M2 conditions and blocked with anti-mouse FcR antibody (CD16/CD32, BD) for 15 min at 4°C in FACS buffer (PBS with 2% FBS and 1 mM EDTA), subsequently cells were stained for 15 min at 4°C with blue-fluorescent reactive dye. Cells were washed three times with FACS buffer, fixed with the eBioscience Fixation/Permeabilization buffer for 40 min at 4°C and washed three times in 1X eBioscience Permeabilization buffer. For intracellular staining, cells were first blocked with anti-mouse FcR antibody (CD16/CD32, BD) in 1X Permeabilization buffer (eBioscience) for 45 min at 4°C prior to staining.

Publication protocol

BMDMs were differentiated for 24 hours in M0, M1 or M2 conditions and blocked with anti-mouse FcR antibody (CD16/CD32, BD) for 15 min at 4°C in FACS buffer (PBS with 2% FBS and 1 mM EDTA), subsequently cells were stained for 15 min at 4°C with blue-fluorescent reactive dye, L23105 (life technologies) to discriminate dead cells, and then surface stained with antibodies for CD11b (clone M1/70, V450 or PE, Biolegend) and CD38 (clone 90, FITC, eBioscience) or isotype control for 15 min at 4°C. Cells were washed three times with FACS buffer, fixed with the eBioscience Fixation/Permeabilization buffer for 40 min at 4°C and washed three times in 1X eBioscience Permeabilization buffer. For intracellular staining, cells were first blocked with anti-mouse FcR antibody (CD16/CD32, BD) in 1X Permeabilization buffer (eBioscience) for 45 min at 4°C prior to staining with anti-Egr2-APC antibody (clone erongr2, eBioscience), anti-Nos2-PE (CXNFT, eBioscience), anti-TNF-α-V650 (MP6-XT22, Biolegend) anti-Arg-1-APC (R and D systems), anti-CD206-Violet 650 (C068C2, Biolegend) or isotype control for 45 min at 4 degrees. After washing 3x in 1X Permeabilization buffer, cells were resuspended in FACS buffer and run on a BD FACSCanto II or BD LSRII Flow Cytometer (BD, NJ). Data were analyzed with FlowJo (Treestar, OR). For cytokine detection, macrophages were incubated in GolgiStop (BD) for 5 hours prior to staining.

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