Neutrophil–platelet (NP)-enriched buffy coats were prepared from heparinised human blood by sedimentation at 37°C. For these experiments buffy coat (30 μL) was added to 970 μL HBSS and incubated for 10 min at 37°C in the presence of the different extracts (50%) or an equal volume of HBSS (0.5 mL, control). This was followed by the addition of ADP (100 μM) or an equal volume of HBSS (background) and incubation for a further 5 min at 37°C. Cells were processed immediately thereafter for flow cytometry. Cell suspensions were stained with 5 μL murine anti-human fluorochrome-labelled monoclonal antibodies directed against CD16–PE cyanin 5 (Beckman Coulter) to detect neutrophils, CD42a–PE (Becton Dickenson) to detect platelets and CD45–Krome Orange (Beckman Coulter) to detect total leukocytes for 15 min at room temperature. This was followed by analysis of the various cell suspensions at a slow flow rate using a Gallios flow cytometer (Beckman Coulter). NP interactions were determined according to the CD16+/CD42a+ coexpression profiles of CD45+ leukocytes and the results expressed as the relative mean fluorescence intensities of CD42a expression of these NP aggregates [20 (link)].
Cd42a pe
Manufactured by BD
Sourced in United States, Germany
CD42a–PE is a fluorescent-conjugated monoclonal antibody used to detect and analyze CD42a, a marker expressed on the surface of platelets. It is a tool commonly used in flow cytometry applications for the identification and enumeration of platelet populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 krome orange, by Beckman Coulter (2 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cd41 percp cy5, by BD (1 mentions)
Cd42b fitc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
12 well plate, by Techno Plastic Products (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Cd61 apc, by BioLegend (1 mentions)
Il 11, by Thermo Fisher Scientific (1 mentions)
Stemspan acf medium, by STEMCELL (1 mentions)
Cd41 apc cy7, by BioLegend (1 mentions)
Apel2 medium, by STEMCELL (1 mentions)
4 protocols using cd42a pe
1
Neutrophil-Platelet Interaction Assay
2
Clofazimine's Impact on Neutrophil-Platelet Interactions
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In this more limited series of experiments, the effects of clofazimine only at fixed concentrations of 2,5 and 5 mg/L on NP interactions were investigated. Buffy coat (20 μL), suspended in 980 μL HBSS, was processed as above for PRP and NP aggregate formation measured following activation with thrombin (0.31 NIH units/mL) or ADP (100 μM) as described previously (Nel et al., 2017 (link)). Following incubation, the cells were stained for 15 min at room temperature in the dark with a cocktail consisting of 5 μL of each of the following, murine, anti-human, fluorochrome-labeled monoclonal antibodies: CD16-allophyocyanin (Biolegend, San Diego, CA, United States), CD42a-PE (Becton Dickenson) and CD45-Krome Orange (Beckman Coulter) to enable detection of neutrophils, platelets and total leukocytes, respectively. This was followed by analysis of the cell suspensions at a slow flow rate using the Gallios flow cytometer. NP heterotypic aggregate formation was determined as CD16+/CD45+ neutrophils co-expressing CD42a. Results are expressed as the relative median fluorescence intensity (MFI) of CD42a as emitted by CD16+/CD45+ neutrophils as an index of the magnitude of the interaction of platelets with individual neutrophils.
3
Platelet Surface GPIX Measurement by Flow Cytometry
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Platelet surface GPIX was measured by whole blood flow cytometry as previously described43 (link),45 (link),71 (link)–73 . Briefly, citrate-anticoagulated whole blood (10 µL) was incubated 15 min RT with an antibody cocktail consisting of CD42a-PE (phycoerythrin, to detect GPIX, catalog # 558819, BD Pharmingen, San Diego, CA), CD42b-FITC (fluorescein isothiocyanate, catalog # 555472, BD Pharmingen) and CD41-PerCP-Cy5.5 (peridinin chlorophyll protein-Cyanine5.5, catalog # 340930, BD Biosciences, San Diego, CA) in the presence of vehicle (10 mM HEPES, 0.15 M NaCl, pH 7.4, “HEPES-saline”), adenosine diphosphate (ADP, catalog # 384, ChronoLog, Havertown, PA) 20 µM, or thrombin receptor activating peptide (TRAP, catalog # 4031274, BaChem, Torrance, CA) 20 µM (total volume 25 µL). Samples were then fixed by addition of 1% formaldehyde in HEPES-saline. Control samples with FITC and PE conjugated normal IgG were used to establish levels of non-specific staining. Samples were analyzed in a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) (threshold on FL3 CD41-PerCP-Cy5.5) and gated for platelet forward and side light scatter. Compensation for fluorescence overlap of the channels was determined using single stained samples.
4
Megakaryocyte Differentiation from Whole Blood
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For MK and platelet experiments, 15–25 ml of whole blood was collected by venipuncture from two healthy controls, P3, P4 and P5 in the presence of 1.6 mg/ml EDTA. As access to patient blood was limited, these experiments were performed once (n = 1), with all samples processed in parallel. MKs were differentiated from whole blood samples using a protocol adapted from Balduini et al.66 (link) Briefly, PBMCs were isolated from using Lymphosep (C C Pro, Oberdorla, Germany). Up to 2 × 106 PBMCs were seeded per well in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) with StemSpan ACF medium (StemCell, Vancouver, Canada) supplemented with 10 ng/ml TPO, FLT3-L, IL-6, and IL-11 (all cytokines from Peprotech, Hamburg, Germany). On day 4, APEL2 medium (StemCell) supplemented with 5% Protein-free Hybridoma Medium II (PFHMII, Gibco by Life Technologies, Darmstadt, Germany) and the same cytokine composition was used. On days 1 or 2, 7, 10, and 14, cell morphology was assessed by microscopy and phenotype was analyzed by flow cytometry. For flow cytometry, cells were blocked with FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with CD42a-PE (BD Biosciences, Heidelberg, Germany), CD41-APC/Cy7, and CD61-APC (both Biolegend, San Diego, USA). Analysis was performed using a FACS Canto II and FACSDiva software v8.0.1 (BD Biosciences).
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