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Fitc conjugated cd14

Manufactured by BD
Sourced in United States

FITC-conjugated CD14 is a fluorescently labeled antibody that binds to the CD14 cell surface receptor. CD14 is a glycosylphosphatidylinositol-anchored protein that serves as a co-receptor for the detection of bacterial lipopolysaccharide.

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3 protocols using fitc conjugated cd14

1

Quantification of Plasma-Derived Microparticles

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Blood was obtained in sodium citrate and processed within 90 min at room temperature. MP counts were performed from fresh samples across the whole study. Samples were centrifuged for 10 min at 150xg and the supernatant, designated platelet-rich plasma (PRP, was then centrifuged at 13000xg for 2 min to pellet the platelets. The resulting supernatant, designated platelet-poor plasma (PPP) contained MP. A 100μl aliquot of PPP was used to assess MP by flow cytometry and the remainder was centrifuged at 17000xg for 45 min to pellet MP. The MP pellet was re-suspended in modified Tyrode’s buffer containing 0.35% BSA and stored at −70°C. MP were analyzed by flow cytometry [Becton-Dickinson LSRI (with upgraded light scatter detectors present on the machine) BD Biosciences] assessing forward and side scatter, and fluorescence using FITC-conjugated annexin V (BD Biosciences) and antibodies specific for endothelial cells ( PE-conjugated CD105 from eBioscience and PE-conjugated CD144 from BD Biosciences ), platelets (FITC-conjugated CD41 from BD Biosciences), leukocytes (PE-conjugated CD18 from BD Biosciences), neutrophils (PE-conjugated CD16b from BD Biosciences) and monocytes (FITC-conjugated CD14 from BD Biosciences). Each marker was counted separately (see supplementary data for gating strategy).
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2

Flow Cytometric Analysis of CD14 and TLR4 Expression

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The cells in each group were collected into FACS tube (352063, BD) and washed in ice-cold phosphate-buffered saline (PBS) twice. THP-1 and PMA-differentiated THP-1 cells were stained with fluorescein isothiocyanate (FITC)-conjugated CD14 (555397, BD Biosciences, Franklin Lakes, NJ, USA) and phycoerythrin (PE)-conjugated TLR4 (564215, BD Biosciences) for 1 h at room temperature. The stained cells were analyzed by flow cytometry (cytoFLEX, A00-1-1102, Beckman Coulter, Brea, CA, USA) and software (CytExpert, Beckman Coulter). For each sample, the count in 10,000 cells was measured. The region of each sample was selected in the forward scatter and side scatter, and then a histogram was used to measure the mean fluorescence intensity of the FITC or PE, which represented the CD14 or TLR4, respectively. FITC-rat IgG2a (BD, 555843) and PE-mouse IgG1 (BD, 559320) were used as isotype controls.
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3

Immunophenotyping of Trophoblast Cells

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TNCs (2 × 105) were labeled with a combination of PerCP‐conjugated CD45 (BD Biosciences, clone 2D1) and APC‐conjugated CD34 (BD Biosciences, clone 8G12) plus: FITC‐conjugated CD14 (BD Biosciences, clone MSE2) for monocyte detection; PhycoErythrin (PE)‐conjugated CD56 (BD Biosciences, clone MY31), FITC‐conjugated CD2 and CD3 (BD Biosciences, clone MOPC21 and UCHT1), FITC‐conjugated CD19 and CD20 (BD Biosciences, clone HIB19 and 2H7) for lymphocyte detection; and FITC‐conjugated CD15 (BD Biosciences, clone MMA) for granulocyte detection. The percentage of cell impurities was calculated as the percentage of CD45+ events after the exclusion of CD34+ events using a FACS Canto II analyzer (BD Biosciences) and FACS DIVA software (Supporting Information Fig. S2).
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