Rabbit polyclonal FITC-conjugated anti-human fibrinogen antibodies (Dako, UK) were used to measure the level of fibrinogen binding as a marker for inside-out signalling to integrin αIIbβ328 (link) and PE-Cy5-conjugated mouse anti-human CD62P antibodies (BD Biosciences, UK) were used to measure the level of P-selectin exposure as a marker for α-granule secretion from platelets1 (link),29 (link). The human PRP was incubated with both the antibodies in HEPES-buffered saline (HBS: 150 mM NaCl, 5 mM KCl, 2 mM MgSO4.7H2O, and 10 mM HEPES, pH 7.4) for various time periods (0, 5, 20, 25, 30 and 50 minutes) with and without different concentrations of LPS chemotypes (LPSEC, LPSSM, and LPSRS). After preincubation, the platelets were exposed to modified Tyrode’s-HEPES buffer (vehicle) or 0.5 μg/mL CRP-XL, 0.25 μg/mL CRP-XL, 10 μM TRAP-6 (Abcam, UK), or 10 μM ADP (Sigma, UK) for various time points at room temperature or 37 °C. The platelets were then fixed using 0.2% (v/v) formyl saline and the level of fluorescence was measured using an Accuri C6 Flow cytometer (BD Biosciences, UK).
Fitc conjugated anti human fibrinogen antibodies
Manufactured by Agilent Technologies
Sourced in United Kingdom
FITC-conjugated anti-human fibrinogen antibodies are laboratory reagents used for the detection and quantification of human fibrinogen in biological samples. These antibodies are labeled with the fluorescent dye FITC (Fluorescein Isothiocyanate), which enables visualization and analysis of fibrinogen-containing samples using fluorescence-based techniques.
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2 protocols using fitc conjugated anti human fibrinogen antibodies
1
Analyzing Platelet Activation Markers
2
Platelet Activation Assays by Flow Cytometry
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The levels of fibrinogen binding and P-selectin exposure were measured by flow cytometry (Accuri C6, BD Biosciences, UK). The platelets (PRP) were treated with different concentrations of isorhapontigenin or resveratrol or a vehicle control for 5 min prior to activation with agonists such as ADP (5 µM), CRP-XL (0.5 µg/ml) or U46619 (1 µM) for further 20 min in the presence of FITCconjugated anti-human fibrinogen antibodies (Dako, UK) and PECy5-conjugated CD62P antibodies (BD Biosciences, UK). The platelets were fixed using 0.2% (v/v) formyl saline and then analysed by flow cytometry by collecting 5000 events within a gated region for platelets. The median fluorescence intensity was used to assess the level of fibrinogen binding (a marker for inside-out signalling to integrin αIIbβ3) and P-selectin exposure (a marker for α-granule secretion) on the platelet surface. The level of fibrinogen binding and P-selectin exposure in the treated samples was calculated by taking the level of median fluorescence intensity obtained with the vehicle control as 100%.
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