The colonies formed in the CFU assays were further differentiated into mature macrophages, in vitro, by culturing the cells in DMEM supplemented with 10% FBS, 10 ng/ml of granulocyte-macrophage colony stimulating factor (GM-CSF), 10 ng/ml macrophage colony stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN) for 4 days. Macrophages were further analyzed by flow cytometry for surface expression of the macrophage cell surface markers, CD14, CD4, and HLA-DR. Macrophages were stained with a phycoerythrin (PE)-conjugated CD4, a PE-conjugated HLA-DR, or a PE-conjugated CD14 antibody (BD Biosciences, San Jose, CA). Flow cytometry was performed on a Beckman Coulter Cytomics FC500 and analyzed with CXP software. Experiments were performed in triplicate.
Pe conjugated cd14 antibody
Manufactured by BD
The PE-conjugated CD14 antibody is a laboratory reagent used for the detection and identification of CD14-positive cells in flow cytometry applications. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein that serves as a co-receptor for the detection of lipopolysaccharide (LPS) and other microbial components. The PE (Phycoerythrin) fluorescent label allows for the visualization and quantification of CD14-expressing cells.
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Lab products found in correlation
Apc conjugated cd11b antibody, by BD (2 mentions)
Pe conjugated cd38 antibody, by BD (2 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Gm csf, by R&D Systems (1 mentions)
M csf, by R&D Systems (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Pe conjugated cd34 antibody, by BD (1 mentions)
Pe cy7 conjugated cd45 antibody, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
1 25 dihydroxyvitamin d3, by Cayman Chemical (1 mentions)
4 protocols using pe conjugated cd14 antibody
1
Macrophage Differentiation and Characterization
2
Flow Cytometric Analysis of Cell Surface Markers
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1×106 cells were collected from cultures and centrifuged at 700 rpm for 5 min. Cell pellets were resuspended in 200 µl 37°C PBS containing 2.5 µl of either APC-conjugated CD11b antibody, PE-conjugated CD38 antibody, or PE-conjugated CD14 antibody (all from BD Biosciences, San Jose, CA). Following 1 h incubation at 37°C, cell surface expression levels were analyzed with a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). APC fluorescence (excitation at 633 nm) was collected with a 660/20 band pass filter and PE fluorescence (excitation at 488 nm) was collected with a 576/26 band pass filter. Undifferentiated control cells were used to determine the fluorescence intensity of cells negative for the respective surface antigen. The gate to determine percent increase of expression was set to exclude 95% of the control population.
3
Human CD34+ Cell Engraftment and Differentiation
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To evaluate the engraftment and multi-lineage differentiation of the human CD34+ cells transduced with the hAS8 lentiviral vector, cells from the peripheral blood, bone marrow, thymus and spleen were analyzed from the transplanted NRG mice. Total cells were obtained from the above-mentioned organs and were labeled with antibodies specific for human immune cells including T cells, B cells, macrophages and CD34+ cells. The following antibodies were used in these studies: T cell antibodies used were a Brilliant violet (BV) 421-conjugated CD3 antibody, an allophycocyanin (APC) H7-conjugated CD4 antibody and an Alexa fluor (AF) 700-conjugated CD8 antibody (BD Biosciences, San Jose, CA). B cell antibodies used were a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD19 antibody (BD Biosciences, San Jose, CA). Macrophage antibodies used were a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD14 antibody (BD Biosciences). CD34+ cell antibodies used were a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD34 antibody (BD Biosciences, San Jose, CA). Flow cytometry was performed using a BD Fortessa and analyzed with CXP software.
4
HL-60 Cell Differentiation and Phenotypic Analysis
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For the phenotypic analysis, cultures of HL‐60 cells were initiated at a density of 0.1 × 106 mL on day 0. The cells were treated with either 1 μmol/L RA (Sigma) or 0.5 μmol/L 1,25‐dihydroxyvitamin D3 (Cayman) to induce differentiation. For the staining of CD38, CD11b, and CD14, 1 × 106 cells were collected and centrifuged at 120 × g for 5 minutes. The cell pellets were resuspended in 200 μL PBS with 2.5 μL of APC‐conjugated CD11b antibody, PE‐conjugated CD38 antibody or PE‐conjugated CD14 antibody (all from BD Biosciences) at 37°C for 1 hour and analyzed with an LSR II flow cytometer (Becton Dickinson). For the cell cycle analysis, the same number of cells was centrifuged at 120 g for 5 min, and stained by resuspension in PI solution (50 mg/mL propidium iodine, 1 ml/mL Triton X‐100, and 1 mg/mL sodium citrate), stored at 4°C overnight, and then analyzed by flow cytometry. Gating was set to exclude debris and doublets.
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