The origin of MPs was detected by flow cytometric analysis. Following incubation of platelet-poor plasma with anti-CD31-PE (5 µl; cat. no. 555027; 1:400; BD Biosciences) and anti-CD41-FITC (5 µl; cat. no. 561849; 1:400; BD Biosciences) for 30 min at 37°C as previously described (16 (link)), flow count calibrator beads (50 µl; Beckman Coulter, Inc.) were added and incubated at 37°C for another 15 min. Platelet-derived MPs [PMPs; CD31(+)/CD41(+)] and endothelial-derived MPs [EMPs; CD31(+)/CD41(−)] were counted and analyzed using an MoFlo XDP Cell Sorter (Beckman Coulter, Inc.; gate size, <1 µm).
Flow count calibrator beads
Manufactured by Beckman Coulter
Sourced in France
Flow Count Calibrator Beads are a reference standard used to calibrate and validate the performance of flow cytometry instruments. They are a suspension of polystyrene beads of known size and concentration, which can be used to align the flow rate and provide a reference for the quantitation of sample events.
Automatically generated - may contain errors
Lab products found in correlation
Moflo xdp, by Beckman Coulter (2 mentions)
Anti cd41 fitc, by BD (1 mentions)
Moflo xdp cell sorter, by Beckman Coulter (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
Coulter cytomic fc 500, by Beckman Coulter (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Flow count beads, by Beckman Coulter (1 mentions)
Anti cd31 fitc antibody, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
8 protocols using flow count calibrator beads
1
Quantification of Platelet and Endothelial Microvesicles
2
Platelet Activation and Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heparin-buffered blood samples were obtained and processed and immediately spun at 13,000 × g for 20 minutes to separate the platelets.
Platelet-poor plasma was incubated for 15 min with a monoclonal antibody against FITC-labeled anti-CD31 antibody (BD Pharmingen. BD Bioscience, CA), followed by incubation with PE-conjugated Annexin V kits according to the manufacturer’s instructions (BD Pharmingen, BD bioscience, CA). The negative control (zero value) was obtained using the isotype antibodies. Flow Count Calibrator beads (Beckman Coulter, Marseille, France) were added. Analyses were performed in a Coulter Cytomic FC 500 flow cytometer (Beckman Coulter) with CXP software (Beckman Coulter). Each sample was measured in triplicate and the mean was taken as the final result.
Platelet-poor plasma was incubated for 15 min with a monoclonal antibody against FITC-labeled anti-CD31 antibody (BD Pharmingen. BD Bioscience, CA), followed by incubation with PE-conjugated Annexin V kits according to the manufacturer’s instructions (BD Pharmingen, BD bioscience, CA). The negative control (zero value) was obtained using the isotype antibodies. Flow Count Calibrator beads (Beckman Coulter, Marseille, France) were added. Analyses were performed in a Coulter Cytomic FC 500 flow cytometer (Beckman Coulter) with CXP software (Beckman Coulter). Each sample was measured in triplicate and the mean was taken as the final result.
3
Endothelial Microparticle Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Platelet-free plasma was obtained by centrifugation at 1,500 rpm for 10 minutes at room temperature, followed by an additional centrifugation at 15,000 rpm for 30 minutes in order to separate the MPs. MPs were resuspended in PBS to determinate the number of endothelial microparticles (EMPs) as described in the following section.
The MPs from plasma were incubated with monoclonal antibody against phycoerythrin (PE)-labeled anti-CD31 (as an endothelial marker; BD Bioscience, San Jose, CA, USA), followed by incubation with fluorescein isothiocyanate–conjugated (FITC) Annexin-V kits according to the manufacturer’s instructions (BD Bioscience). The negative control was obtained using the anti-isotype antibodies. An equal volume of flow count calibrator beads (Beckman Coulter, Marseilles, France) were added. Fluorescence-activated cell sorter analysis was performed in an Accuri C6 flow cytometer (BD Bioscience).
The MPs from plasma were incubated with monoclonal antibody against phycoerythrin (PE)-labeled anti-CD31 (as an endothelial marker; BD Bioscience, San Jose, CA, USA), followed by incubation with fluorescein isothiocyanate–conjugated (FITC) Annexin-V kits according to the manufacturer’s instructions (BD Bioscience). The negative control was obtained using the anti-isotype antibodies. An equal volume of flow count calibrator beads (Beckman Coulter, Marseilles, France) were added. Fluorescence-activated cell sorter analysis was performed in an Accuri C6 flow cytometer (BD Bioscience).
4
Quantification of Endothelial Microparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture supernatants from endothelial cells treated with native and oxidized albumin for 24 hours were collected and cleared from detached cells and cell fragments by centrifugation at 1,500 rpm for 10 minutes at room temperature. The supernatants were then subjected to centrifugation at 15,000 rpm for 30 minutes. Pelleted EMPs were resuspended in PBS and then quantified. An equal volume of flow count calibrator beads (Beckman Coulter) was added. The EMPs in HUVEC were determined and quantified in an Accuri C6 flow cytometer (BD Bioscience) using forward scatter intensity value (size) and side scatter intensity value (granularity).
5
Quantification of Endothelial Microparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All patients preoperatively fasted overnight as did healthy volunteers. Blood samples were drawn and centrifuged to obtain platelet-poor plasma (PPP) [4 (link)]. 50 μL of PPP was incubated with 4 μl of anti-CD31-PE and 4 μL of anti-CD42b-FITC antibodies at room temperature for 20 min with gentle orbital shaking in the dark [4 (link)]. The samples incubated with corresponding isotype control (all from Beckman Coulter, France) were used as controls. After labeling, samples were analyzed via MoFlo XDP (Beckman coulter) by an independent examiner who was blinded to study arm and intentions. Before analysis, 50 μL flow count calibrator beads (Beckman Coulter) with known concentration provided by the manufacturer were added into the antibody-labeled tubes. After excluding non-specific fluorescence, those positively labeled by anti-CD31-PE and negatively labeled by anti-CD42b-FITC and <1 μm in size were considered to be EMPs [4 (link)].
6
Quantification of Apoptotic Microparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were centrifuged at 3000 rpm for 10 min within 2 h from collection, and serum was immediately stored in aliquots at 280°C until analysis as follows: Fifty microliters Platelet‐poor plasma heparin was incubated with a monoclonal antibody anti‐CD31‐FITC antibody (BD Pharmingen. BD Bioscience, CA, USA), and anti‐CD41‐Pacific blue, followed by 20 min incubation with PE‐conjugated Annexin V (AV) kits according to the manufacturer's instructions (BD Pharmingen, BD bioscience, CA, USA). AV+ was used to determine apoptotic microparticles, CD31 FITC and CD41‐Pacifiblue were used to differentiate between CD31 + CD41 + PMPs and CD31 + CD41 – EMPs. The negative control (zero value) was obtained using the isotype antibodies. Flow Count Beads (Beckman Coulter, Marseille, France) were added. MPs were identified as events with a .1–1 μm diameter on forward light scatter (FSC) and side‐angle light scatters (SSC) intensity dot plot representation, by comparison to flow cytometry calibration beads (Flow count ® calibrator beads, Beckman Coulter, Marseille, France). Microparticles were analyzed by flow cytometry in duplicate (BD LSRFortessa; BD Biosciences, San Diego, CA, USA). Data represent the mean (± SEM) of two independent experiments.
7
Quantification of Endothelial Microparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The methods have been already described. 24, 25 Citrated blood (6 ml) was drawn from the cubital vein contralateral to intervention arm and processed within 2 h. Platelet-rich plasma was obtained by centrifugation of whole blood at 300g over 15 min at room temperature. Platelet-free plasma was obtained by 2 successive centrifugations of platelet-rich plasma at 10 000g for 5 min at room temperature. Briefly, samples were incubated for 30 min with fluorochrome-labeled antibodies or matching isotype controls and analyzed in a Canto II flow cytometer (Beckton Dickinson, Heidelberg, Germany). Microbead standards (1.0 µm) were used to define MPs as <1 µm in diameter. The EMP subpopulations were defined as CD31 + / CD41 -, CD62e + , or CD144 + events. The total number of EMP was quantified using flow-count calibrator beads (Beckman Coulter; 20 ml).
8
Quantifying Annexin V-Positive Microparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The platelet-poor plasma was thawed on ice, and 10 ml was incubated with annexin V that had been previously diluted in 90 ml of annexin V binding buffer ( 2X, containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 0.28 M sodium chloride, and 5 mM calcium chloride; pH 7.5), for 30 minutes, at room temperature, in the dark. Platelet-poor plasma incubated with annexin V in 2X binding buffer, containing 5 mM EDTA (ethylenediaminetetraacetic acid) but no calcium chloride, was used as a negative control. Before analysis, 10 ml of flow count calibrator beads (Beckman Coulter Inc, Fullerton, Calif) with known concentration were added into the samples. The annexin V binding MPs were analyzed in MoFlo XDP (Beckman Coulter Inc). After exclusion of background noise, those positively labeled for annexin V in the gate (size ¼ 1 mm) were considered to be MPs.
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!