Blood from healthy mice was drawn via cardiac puncture with a heparinised syringe. The blood was diluted 1:2 with PBS and immediately stained with fluorescent antibodies (CD41-BV421, clone MWReg30, BioLegend; CD45-PE, clone 30-F11, BioLegend; Ly-6B.2-FITC, clone 7/4, Bio-Rad) in 1:200 dilution. Samples were stimulated simultaneously with 1 µM ADP (Sigma-Aldrich) where indicated. After 20 min, the reaction was stopped by fixing the samples in 2% PFA (Roth) and 15 min later diluted again 1:10 in PBS and subsequently analysed via imaging flow cytometry using the AMNIS® ImageStream® II (Luminex Corporation, Austin, TX, USA).
Cd41 bv421 clone mwreg30
Manufactured by BioLegend
CD41-BV421 (clone MWReg30) is a fluorochrome-conjugated antibody used for the detection and quantification of the CD41 antigen, also known as the platelet glycoprotein IIb (GPIIb) subunit. This antibody is designed for flow cytometric applications.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine diphosphate (adp), by Merck Group (2 mentions)
Cd45 pe clone 30 f11, by BioLegend (2 mentions)
Paraformaldehyde (pfa), by Carl Roth (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Hepes, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BioLegend (1 mentions)
Xylazine, by Bayer (1 mentions)
Cd11b pe clone m1 70, by BD (1 mentions)
Cd45 apc cy7 clone 30 f11, by BioLegend (1 mentions)
4 protocols using cd41 bv421 clone mwreg30
1
Platelet Activation Assay in Mice
2
TLR4/MD2 Expression and Platelet Activation
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For the analysis of TLR4/MD2 expression on murine platelets, platelets were isolated from TLR4PF4Cre- and TLR4PF4Cre+ mice via repetitive slow-speed centrifugation and stained with CD41-BV421 (clone MWReg30, BioLegend) and TLR4/MD2-APC (clone MTS510, Thermo Fisher Scientific) antibodies for 20 min at RT. Expression levels were investigated in a FACSCantoII flow cytometer (BD Bioscience). Stimulation of isolated WT platelets with S100A8/A9 tetramers was performed according to Colicchia et al. with minor modifications [28 (link)]. Murine platelets were isolated via repetitive slow-speed centrifugation and resuspended in Tyrode’s buffer (20 mM HEPES, 136 mM NaCl, 2.6 mM KCl, 12 mM NaHCO3, 5.5 mM D-glucose, 2 mM CaCl2, 1 mM MgCl2). Platelets (2 × 106) were stimulated with 40 µg/mL S100A8/A9 (produced and purified by T. Vogl). CD62P mobilisation and GPIIbIIIa activation were assessed by antibody staining (CD62P-PE, clone RMP-1, BioLegend; act. GPIIbIIIa-PE, clone JON/A, Emfret Analytics) and analysed with FlowJo software.
3
Platelet-Neutrophil Interaction Assay
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Platelets and bone marrow neutrophils were isolated as described above. Platelets and neutrophils were combined at a 10:1 ratio in a final volume of 100 µL RPMI (PAN) containing 0.5% FCS (PAN) and 25 mM HEPES (Sigma). The cells were stimulated with 10 µM fMLP (Sigma) and 0.1 U/mL thrombin (Sigma) for 10 min at RT with subsequent antibody staining (CD41-BV421 (clone MWReg30, BioLegend), Gr-1-AF633 (clone RB6-8C5, purified from hybridoma supernatant), CD45-PE (clone 30-F11, BioLegend), Ly6B.2-FITC (clone 7/4, Bio-Rad)). CD41+ signals were compared to isotype controls (BV421 Rat IgG1, κ isotype ctrl antibody, BioLegend) and measurements were performed with a FACSCantoII flow cytometer.
4
Murine Whole Blood Protein Expression
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Surface expression of proteins on neutrophils and platelets was assessed in murine whole blood. Mice were anaesthetised via intraperitoneal injection of ketamine (125 µg/g body weight; WDT) and xylazine (12.5 µg/g body weight; Bayer, Leverkusen, Germany) and blood was drawn via cardiac puncture into an ACD (Sigma-Aldrich)-coated syringe with a 27G cannula. Blood was diluted 1:5 with PBS. As a low stimulus, 1 µM ADP (Sigma-Aldrich) was added, while controls were left untreated. Diluted full blood was stained with CD45-APC/Cy7 (clone 30-F11, BioLegend), Ly-6G-AF488 (clone 1A8, BioLegend), CD41-BV421 (clone MWReg30, BioLegend), CD11a-PE (clone M7/14, BioLegend), CD11b-PE (clone M1/70, BD Bioscience), CD162-PE (clone 2PH1, BD Bioscience), CD40-PE (clone FGK45, BioLegend), GP1bα-AF488 (clone Xia.G5, Emfret Analytics, Würzburg, Germany), Ly49Q mAb-PE (clone JON/A, emfret), CD154-PE (clone MR1, BioLegend), CD62P-PE (clone RMP-1, BioLegend) and CD102-AF488 (clone 3C4, BioLegend). Neutrophils were considered CD45 + Ly-6G+ populations, while platelets were identified as CD45-CD41+.
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