pHrodo Red-labeled MT4CCR5 and MT4CCR5/NL4-3 cells 1, 2, 3, and 4 days post-infection were cultured with macrophages in the absence and presence of Gas6 (1 μg/ml) for 90 min. Phagocytosis was analyzed by flow cytometry as previously described. MT4CCR5/NL4-3 and MT4CCR5/Ada cells (1×105 cells) at 2 days post-infection were co-cultured with various numbers of macrophages (0 – 4×105 cells) in the absence and presence of Gas6 (1 μg/ml) for 15 h. The supernatants of the cells were filtered and subjected to titration of HIV-1 using GHOST (3) CXCR4+CCR5+ cells. To investigate viability of cells engulfed in macrophages, MT4CCR5/NL4-3 cells at 2 days post-infection were labeled by CellTrace Violet and Ghost Red Dye 780. The labeled cells were co-cultured with macrophages for 90 min. The cells were stained by PE-conjugated anti-CD14 antibody (BD Biosciences), followed by analysis by imaging flow cytometry. The internalization of CellTrace Violet signal into CD14-positive masks were first analyzed. The cells above internalization score 1.5 were analyzed for the signals of Ghost Red Dye 780 staining.
Pe conjugated anti cd14 antibody
Manufactured by BD
PE-conjugated anti-CD14 antibody is a monoclonal antibody that specifically binds to the CD14 protein expressed on the surface of monocytes and macrophages. It is conjugated with the fluorescent dye phycoerythrin (PE) to enable detection and analysis of CD14-positive cells using flow cytometry.
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3 protocols using pe conjugated anti cd14 antibody
1
Macrophage-Mediated Clearance of HIV-Infected Cells
2
Macrophage-Mediated Clearance of HIV-Infected Cells
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pHrodo Red-labeled MT4CCR5 and MT4CCR5/NL4-3 cells 1, 2, 3, and 4 days post-infection were cultured with macrophages in the absence and presence of Gas6 (1 μg/ml) for 90 min. Phagocytosis was analyzed by flow cytometry as previously described. MT4CCR5/NL4-3 and MT4CCR5/Ada cells (1×105 cells) at 2 days post-infection were co-cultured with various numbers of macrophages (0 – 4×105 cells) in the absence and presence of Gas6 (1 μg/ml) for 15 h. The supernatants of the cells were filtered and subjected to titration of HIV-1 using GHOST (3) CXCR4+CCR5+ cells. To investigate viability of cells engulfed in macrophages, MT4CCR5/NL4-3 cells at 2 days post-infection were labeled by CellTrace Violet and Ghost Red Dye 780. The labeled cells were co-cultured with macrophages for 90 min. The cells were stained by PE-conjugated anti-CD14 antibody (BD Biosciences), followed by analysis by imaging flow cytometry. The internalization of CellTrace Violet signal into CD14-positive masks were first analyzed. The cells above internalization score 1.5 were analyzed for the signals of Ghost Red Dye 780 staining.
3
Isolation and Culture of Human Monocyte-Derived Macrophages
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Twenty milliliters of participants’ peripheral blood samples were collected and inserted in tubes containing ethylenediaminetetraacetic acid (EDTA). Within five hours of the time of collection, samples were diluted at 1:2 in phosphate-buffered saline (GIBCO Invitrogen) at a pH of 7.2. Ficoll (Lymphodex, Inno-Train) density gradient centrifugation was used to extract peripheral blood mononuclear cells (PBMCs), which were then washed in PBS. Cell sorter columns were used to separate monocytes after they were treated with MACS CD14 microbeads to undergo positive CD14 selection (all from Miltenyi Biotec). Immunofluorescence labeling of the separated monocytes was done with a phycoerythrin (PE)-conjugated anti-CD14 antibody (BD bioscience) and flow cytometry results revealed a purity of 92–95% [20 (link)]. Following that, the isolated CD14 positive monocytes were cultured in the Roswell Park Memorial Institute (RPMI) at a concentration of 500,000 cells per well in 24-well plates. The media contained 2 mM L-glutamine (Biosera), 10% fetal bovine serum (FBS; Gibco BRL), 0.1 mg/ml streptomycin, and 100 U/ml penicillin (Sigma). For seven days, 0.05 µg/ml recombinant macrophage-colony stimulating factor (M-CSF; eBioscience) was added to culture media to convert monocytes into macrophages [24 (link)].
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