Dihydrorhodamine-123 (DHR-123), Hoechst 33342 and Sytox Green nuclear dyes were purchased from Molecular Probes (Eugene, OR) and stored at −20°C. Phorbyl 12-myristate 13-acetate (PMA) and Diphenyleneiodonium chloride (DPI) were purchased from Sigma Aldrich (Milwaukee, WI). PMA was dissolved in DMSO at a stock solution concentration of 1.6mM and stored at −20°C. DPI was dissolved in de-ionized water at a concentration of 10mM, and stored at room temperature. Recombinant human P-selectin (R&D Systems, Minneapolis, MN) was re-suspended in deionized water (100 μg/mL) and stored at 4°C. Phycoerythrin conjugated anti-human CD14 (Clone 61D3) was purchased from eBioScience (San Diego, CA). AlexaFluor 647 conjugated anti-mouse/human CD11b conjugated (Clone M1/7) and AlexaFluor 790 conjugated anti-human CD45 (Clone H130) were purchased from BioLegend (San Diego, CA).
Recombinant human p selectin
Manufactured by R&D Systems
Sourced in France
Recombinant human P-selectin is a cell adhesion molecule that plays a role in the initial tethering and rolling of leukocytes on vascular endothelium. It is a transmembrane glycoprotein that is expressed on the surface of activated endothelial cells and activated platelets.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using recombinant human p selectin
1
Immune Cell Labeling and Activation Protocol
2
Nanoparticle Binding Kinetics under Flow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the interaction of unloaded NPs with their molecular target, an in vitro binding assay under flow was developed [42] . Micro-channels of Vena8 Fluoro + chambers (Cellix Ltd, Dublin, Ireland) were coated overnight with recombinant human P-selectin (R&D systems France, Lille, France) at 50 µg/mL and rinsed with NaCl 0.9% just before use.
NPs diluted at 1 mg/mL in NaCl 0.9% were perfused through channels for 5 minutes either at venous or arterial flow with an ExiGo™ pump (Cellix Ltd, Dublin, Ireland). Venous and arterial conditions corresponded to a shear stress of 6.75 dyne/cm 2 and of 67.5 dyne/cm 2 , respectively.
The binding of NPs was visualized in real time under fluorescence microscopy (Axio Observer, Carl Zeiss Microimaging GmbH, Iena, Germany). The quantitative analysis was limited to NP clusters that formed onto the coated channel because of the resolving power of the fluorescence microscope. After rinsing with NaCl 0.9%, 10 fields per channel (area of 1230 µm x 105 µm) were imaged and analyzed with the image analysis software ImageJ (NIH, Bethesda, U.S.) to quantify the number of fluorescent NPs clusters. Unspecific binding was controlled on uncoated channels and on channels coated with Bovine Serum Albumin at 50 µg/mL.
NPs diluted at 1 mg/mL in NaCl 0.9% were perfused through channels for 5 minutes either at venous or arterial flow with an ExiGo™ pump (Cellix Ltd, Dublin, Ireland). Venous and arterial conditions corresponded to a shear stress of 6.75 dyne/cm 2 and of 67.5 dyne/cm 2 , respectively.
The binding of NPs was visualized in real time under fluorescence microscopy (Axio Observer, Carl Zeiss Microimaging GmbH, Iena, Germany). The quantitative analysis was limited to NP clusters that formed onto the coated channel because of the resolving power of the fluorescence microscope. After rinsing with NaCl 0.9%, 10 fields per channel (area of 1230 µm x 105 µm) were imaged and analyzed with the image analysis software ImageJ (NIH, Bethesda, U.S.) to quantify the number of fluorescent NPs clusters. Unspecific binding was controlled on uncoated channels and on channels coated with Bovine Serum Albumin at 50 µg/mL.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!