GFP expressing LR73 fibroblasts were seeded in a 24-well plate at a density of 0.2×105 cells per well. Next day, cells were treated with Lipofectamine 2000 (Thermo Fisher) with specific siRNAs according to the manufacturer’s protocol, two days prior to efferocytosis assays. Thymocytes were isolated from 4 to 6-week-old mice and treated with 25 μM dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 10 μM TAMRA in serum-free HBSS for 45 min at 37 °C, washed, and incubated in serum-containing assay media for an additional 25min. Phagocytes were pre-incubated with Cytochalasin D (Sigma-Aldrich, 1 μM) for 1 h and, in some experiments, Tritbutyltin chloride (Sigma; 10 μM for 6 h), or with RGDS peptide for 30 min (Tocris 100 μM); in some cases apoptotic cells were incubated with Annexin V (Abcam, 10 μg/ml for 30 min). Apoptotic cells were subsequently co-cultured with phagocytes, in the presence of vehicle or indicated inhibitor, at a 1:10 phagocyte to target ratio for 30 minutes. After the incubation, apoptotic cells were removed via three cold PBS washes and phagocytes were harvested via trypsin/EDTA and assessed for the percentage and MFI of GFP+ TAMRA+ LR73 cells by flow cytometry.
Tritbutyltin chloride
Manufactured by Merck Group
Tritbutyltin chloride is an organotin compound with the chemical formula (C₄H₉)₃SnCl. It is a clear, colorless liquid used as an intermediate in the production of other tin-based compounds. The core function of tritbutyltin chloride is to serve as a precursor for the synthesis of a variety of organotin materials.
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2 protocols using tritbutyltin chloride
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Efferocytosis Assay of Apoptotic Cells
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Efferocytosis Assay of Apoptotic Cells
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GFP expressing LR73 fibroblasts were seeded in a 24-well plate at a density of 0.2×105 cells per well. Next day, cells were treated with Lipofectamine 2000 (Thermo Fisher) with specific siRNAs according to the manufacturer’s protocol, two days prior to efferocytosis assays. Thymocytes were isolated from 4 to 6-week-old mice and treated with 25 μM dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 10 μM TAMRA in serum-free HBSS for 45 min at 37 °C, washed, and incubated in serum-containing assay media for an additional 25min. Phagocytes were pre-incubated with Cytochalasin D (Sigma-Aldrich, 1 μM) for 1 h and, in some experiments, Tritbutyltin chloride (Sigma; 10 μM for 6 h), or with RGDS peptide for 30 min (Tocris 100 μM); in some cases apoptotic cells were incubated with Annexin V (Abcam, 10 μg/ml for 30 min). Apoptotic cells were subsequently co-cultured with phagocytes, in the presence of vehicle or indicated inhibitor, at a 1:10 phagocyte to target ratio for 30 minutes. After the incubation, apoptotic cells were removed via three cold PBS washes and phagocytes were harvested via trypsin/EDTA and assessed for the percentage and MFI of GFP+ TAMRA+ LR73 cells by flow cytometry.
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