bmdm culture medium  (PeproTech)

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Macrophage-derived exosomes promote neutrophil necroptosis following hemorrhagic shock

doi: 10.1189/jlb.3HI0517-173R

Figure Lengend Snippet: (A) WT mice were administered with clodrosome or control vehicle encapsome (both 150 µl/mouse, i.t.), 24 h before HS Exosomes were isolated from BALF and CD63 (marker of exosomes) measured by flow cytometry. MFI, Mean fluorescence intensity. (B) Mice were subjected to HS or sham operation and exosomes isolated from BALF and analyzed by transmission electron microscopy for overall morphology and numbers. (C) BMDMs were subjected to normoxia (20% O2) or hypoxia (1% O2) for 15 h, followed by reoxygenation (20% O2) for 5 h (Hypo/Reox). Exosomes were isolated from culture medium and detected by CD63 expression by flow cytometry. (D) PMN cocultured with or without BMDM under normoxia or hypoxia-reoxygenation, with or without DMA (25 𝜇M). PMN death was analyzed by flow cytometry with staining of FITC-Ly6G, PE-Annexin V, and 7-AAD. (E) Immunofluorescence images showing colocalization of RIPK1 (green) and RIPK3 (red) in PMN, cocultured with or without BMDM under hypoxia-reoxygenation conditions, with DMA (25 𝜇M) or vehicle (DMSO). (F) PMNs were incubated the exosomes from the following: 1) normoxia BMDM culture medium; 2) hypoxia-reoxygenation BMDM culture medium; 3) hypoxia-reoxygenation BMDM accompanied with 30 𝜇M Nec-1 culture medium; and 4) hypoxia-reoxygenation BMDM, excluding exosome culture medium for 20 h. PMN cell death was detected by flow cytometry with staining of FITC-Ly6G, PE-Annexin V, and 7-AAD. (G) Immunofluorescence images showing PMNs incubated with Dil-labeled M𝜙-derived exosomes (red) for 2 h. Nuclei were counterstained with Hoechst (blue). All results are representative of 3 independent experiments. The images are counted by 3 random fields. The graphs show means ± SEM, n = 3; *P < 0.05; **P < 0.01 compared with other groups

Article Snippet: The resultant cells were suspended in BMDM culture medium (DMEM containing 10% FBS, supplemented with 50 μ g/ml penicillin/streptomycin and 10 ng/ml recombinant M-CSF; Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 10 6 cells/ml and seeded into 6-cm plates.

Techniques: Control, Isolation, Marker, Flow Cytometry, Fluorescence, Transmission Assay, Electron Microscopy, Expressing, Staining, Immunofluorescence, Incubation, Labeling, Derivative Assay

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Macrophage-derived exosomes promote neutrophil necroptosis following hemorrhagic shock

doi: 10.1189/jlb.3HI0517-173R

Figure Lengend Snippet: (A) PMN cocultured with or without BMDM under normoxia (20% O2) or hypoxia (1% O2) for 15 h followed by reoxygenation (20% O2) for 5 h (Hypo/Reox). PMNs were stained with PE-CD11b (marker of PMN) and CM-H2DCFDA (ROS) and analyzed by flow cytometry. (B) PMN cocultured with or without BMDM accompanied with/without NAC (50 µg/ml) under normoxia or hypoxia-reoxygenation. PMN cell death was analyzed by flow cytometry with staining of FITC-Ly6G, PE-Annexin V, and 7-AAD. (C) PMN cocultured with or without BMDM under normoxia or hypoxia-reoxygenation with/without DMA (25 µM) or vehicle (DMSO). PMNs were stained with PE-CD11b (PMN marker) and CM-H2DCFDA (ROS) and levels measured by flow cytometry. (D) PMNs were incubated for 20 h with exosomes isolated from the following: 1) normoxia BMDM culture medium; 2) hypoxia-reoxygenation BMDM culture medium; and 3) hypoxia-reoxygenation BMDM, excluding exosome culture medium. The PMN ROS expression was measured by flow cytometry with staining of PE-CD11b (gating for PMN population) and CM-H2DCFDA (ROS). All results are representative of 3 independent experiments. The graphs show means ± SEM, n = 3; **P < 0.01 compared with other groups

Article Snippet: The resultant cells were suspended in BMDM culture medium (DMEM containing 10% FBS, supplemented with 50 μ g/ml penicillin/streptomycin and 10 ng/ml recombinant M-CSF; Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 10 6 cells/ml and seeded into 6-cm plates.

Techniques: Staining, Marker, Flow Cytometry, Incubation, Isolation, Expressing