Flow Cytometry Protocol for Cell Sorting

Flow cytometry was performed as described (21 (link)) using anti-mouse CD16/32 (Fc Block; BD Biosciences) before staining with primary antibody or isotype controls. Cells were surface-stained, then fixed/permeabilized (Fix-Perm buffer, eBioscience) before intracellular staining. Antibodies are listed in Table 2. Uptake of low-density lipoproteins was assessed by incubating PEC with BODIPY-labeled LDL (10 µg/ml, Invitrogen) (16 (link)). Data were acquired and analyzed as above. CD11b⁺Ly6C^hi LyG^- and CD11b⁺CD138⁺ Ly6G^-cells were sorted using a FACSaria cell sorter and 40,000 cells/subset were lysed immediately for RNA extraction.

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Han S., Zhuang H., Shumyak S., Wu J., Xie C., Li H., Yang L.J, & Reeves W.H. (2018). Liver X Receptor Agonist Therapy Prevents Diffuse Alveolar Hemorrhage in Murine Lupus by Repolarizing Macrophages. Frontiers in Immunology, 9, 135.

Publication 2018

anti-mouse CD16/32 (Fc Block; Fix-Perm buffer BODIPY-labeled LDL

Antibodies Antibody Bodipy Buffer Cd11b Cd138 Cells Flow cytometry Intracellular Isotype Low density lipoproteins Mouse Perm

Corresponding Organization :

Other organizations : University of Florida

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Uptake of low-density lipoproteins assessed by incubating PEC with BODIPY-labeled LDL (10 µg/ml, Invitrogen)

dependent variables

CD11b+Ly6Chi LyG- and CD11b+CD138+ Ly6G- cells were sorted using a FACSaria cell sorter and 40,000 cells/subset were lysed immediately for RNA extraction

control variables

Flow cytometry was performed as described (21 (link)) using anti-mouse CD16/32 (Fc Block; BD Biosciences) before staining with primary antibody or isotype controls
Cells were surface-stained, then fixed/permeabilized (Fix-Perm buffer, eBioscience) before intracellular staining

positive controls

Primary antibody

negative controls

Isotype controls

Annotations

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