Plasma samples were stained with the following antibodies: CD41a-FITC (BD Pharmingen), annexinV-PE (BioLegend, San Diego, CA, USA), CD45-BrilliantViolet421 (BioLegend), and CD144-PE/Cy7 (BioLegend) (1 µL of each per 50 µL of plasma) for 15 min at 37 °C, then fixed with PFA and diluted with PBS directly before the measurement. PFA and PBS were filtered through a 0.1 µm filter to reduce the background. Samples were measured on a NovoCyte Quanteon Flow Cytometer (Agilent, Santa Clara, CA, USA), flow rate slow, with no gating, for a fixed amount of time. Obtained data are presented as either densities (events/µL) or median florescence intensity (MFI). A flow cytometry schema of gating platelet-derived and procoagulant microvesicles is shown in Figure 1 A.
Cd41a fitc
Manufactured by BD
Sourced in United States
The CD41a-FITC is a laboratory instrument used for the detection and quantification of the CD41a protein marker on the surface of cells. It utilizes fluorescein isothiocyanate (FITC) as the labeling dye to enable the visualization and analysis of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 pe, by BioLegend (1 mentions)
Novocyte quanteon flow cytometer, by Agilent Technologies (1 mentions)
Cd14 apc, by BD (1 mentions)
Cd62l pe, by BD (1 mentions)
Cd45 v450, by BD (1 mentions)
Cd71 fitc, by Thermo Fisher Scientific (1 mentions)
Cd34 pe, by Thermo Fisher Scientific (1 mentions)
Cd43 apc, by Thermo Fisher Scientific (1 mentions)
Cd144 pe, by Beckman Coulter (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Igg1 fitc, by BD (1 mentions)
Fitc conjugated anti annexin 5 antibody, by BD (1 mentions)
Igg1 percp, by BD (1 mentions)
Flow count fluorosphere, by Beckman Coulter (1 mentions)
Cd45 percp, by BD (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
6 protocols using cd41a fitc
1
Flow Cytometric Analysis of Plasma Microvesicles
2
Hydrogel-Mediated Complement Inhibition
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The amount of Atto488 released from the hydrogel as a model drug was determined by fluorescence measurement at λex. = 485 nm/λem. = 535 nm against an Atto488 standard. The inhibitory effect of soluble as well as hydrogel‐released compstatin 4W9A was tested in whole blood, where the complement system was activated with 10 µg mL−1 nonopsonized zymosan for 30 min at 37 °C. The supernatant of cleaved PMX53 conjugated hydrogels was exposed to isolated granulocytes in the presence of 5% zymosan‐activated serum for 20 min at 37 °C. Cell activation was determined by flow cytometry using CD11b‐VioBlue staining (Biolegend) for isolated granulocytes and additionally CD14‐APC and CD41a‐FITC (both BD Biosciences, New Jersey, USA) for whole blood samples. Leukocyte elastase and C5a concentrations were determined using the commercial ELISA kits Hycult human elastase ELISA (Hycult Biotech, Uden, Netherlands) and C5a micro ELISA (DRG, Marburg, Germany) according to the manufactures instructions.
3
Quantifying Platelet-Leukocyte Aggregates and Inflammatory Markers
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Blood samples for PLA and TNF-α were collected after anesthesia induction. Samples were incubated with CD62L-PE (BD, Basel, Switzerland), CD41a-FITC (BD) and DRAQ5 (Biotium, Hayward, CA, USA). An IgG1-FITC/PE antibody (BD) served as an isotype control. PLA was identified using CD62L/CD41a dot plots after confirming cell nuclei using DRAQ5. Putative PLA was verified by sorting stained samples using FACSAria and observation under a confocal fluorescence microscope (Supplementary Figure). Details of PLA determination are provided in Supplementary Information 4 .
For analysis of plasma TNF-α, arterial blood was centrifuged at 4 °C for 15 min at 1000 g. TNF-α concentration was determined using a commercial enzyme-linked immunosorbent assay kit (High Sensitivity Kit, R&D Systems, Minneapolis, MN, USA) with a detection limit of 0.038 pg/ml.
For analysis of plasma TNF-α, arterial blood was centrifuged at 4 °C for 15 min at 1000 g. TNF-α concentration was determined using a commercial enzyme-linked immunosorbent assay kit (High Sensitivity Kit, R&D Systems, Minneapolis, MN, USA) with a detection limit of 0.038 pg/ml.
4
Platelet Viability Determination
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Platelet samples were transferred to tubes containing K3 EDTA. CD41a FITC (BD Biosciences, San Jose, CA, USA) and 7-aminoactinomycin D (7-AAD) were used to determine viable platelets. The incubated cells were analyzed using the FACSDiva software for FACSCanto II model flow cytometry (BD Biosciences).
5
Flow Cytometry Analysis of Hematopoietic Cells
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The cells were harvested, and 2 × 105 cells were placed in aliquots in phosphate-buffered saline (PBS) containing 1% BSA (PBS/BSA) and centrifuged (200g) for 5 minutes. Antibodies were added directly to cell pellets that were then resuspended to a final volume of 100 μl, incubated for 30 minutes on ice, washed in 5 ml PBS/BSA, and resuspended in 300 μl. The samples were analyzed using an LSRFortessa (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com ) using FACSDiva acquisition (BD Biosciences) and FlowJo analysis software (FlowJo, Ashland, OR, http://www.flowjo.com ), or sorted on a FACSARIA (BD Biosciences). Anti-human antibodies included CD71-FITC (catalog no. 11-0719; eBioscience, San Diego, CA, http://www.ebioscience.com ), CD43-APC (catalog no. 17-0439-42; eBioscience), glycophorinA (M) (CD235a), efluor450 (catalog no. 48-9884; eBioscience), CD34-PE (catalog no. 12-0349; eBioscience), CD41a-FITC (BD Biosciences), CD45-V450 (BD Biosciences), and CD144-PE (Beckman Coulter, Brea, CA, http://www.beckmancoulter.com ). Mouse IgG1 APC isotype control (catalog no. 17-4714-81; eBioscience) and mouse IgG1 PE isotype control (catalog no. 12-4714-81; eBioscience) were used (supplemental online Fig. 2 ).
6
Multiplex Flow Cytometry for Plasma Microparticle Analysis
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