Flow cytometry analyses were performed using FACSCalibur (Becton Dickinson, Heidelberg, Germany) [40 (link)]. Diluted PRP aliquots (5 × 107 platelets/mL) were fixed and stained with FITC-labeled monoclonal surface antibody against CD41 (GPIIb/IIIa-complex), CD42a (GPIb/IX) and CD42b (GPIb) (Coulter, Immunotech, Marseille, France). FITC-labeled anti-VWF (Bio-Rad AbD Serorech, Puchheim, Germany) and Alexa Fluor 488-labeled anti-fibrinogen (Invitrogen, Waltham, MA USA) were used to stain the platelets. In the presence of 1.25 mM Gly Pro-Arg-Pro (Bachem, Bubendorf, Switzerland) diluted PRP (5 × 107 platelets/mL) was stimulated with several concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5 and 1 U/mL; Siemens Healthineers, Marburg, Germany) to conduct the CD62 and CD63 expression analyses. The platelets were also stained with monoclonal FITC-labeled anti-CD62 (P-selectin) and anti-CD63 antibodies (lysosomal membrane associated glycoprotein 3, LAMP-3; Immunotech, Marseille, France).
Gly pro arg pro
Manufactured by Bachem
Sourced in Switzerland
Gly-Pro-Arg-Pro is a tetrapeptide with the amino acid sequence glycine-proline-arginine-proline. It is a synthetic compound commonly used in various research and laboratory applications, such as biochemical assays and cell-based experiments. The core function of Gly-Pro-Arg-Pro is to serve as a tool for researchers to study biological processes and test hypotheses, without interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin, by Siemens (4 mentions)
Alexa fluor 488 labeled anti fibrinogen, by Thermo Fisher Scientific (4 mentions)
Facscalibur, by BD (4 mentions)
Fitc labeled anti vwf, by Bio-Rad (4 mentions)
Thrombin, by Bachem (2 mentions)
Anti cd62p pe, by BD (2 mentions)
Accuri c6 plus flow cytometer, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
7 protocols using gly pro arg pro
1
Flow Cytometry Analysis of Platelet Activation
2
Platelet Granule Release in M Protein Stimulation
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Platelet granule release in response to the different M proteins was investigated using ELISA. PRP was prepared from five healthy donors (donors 1 to 5) as previously described and stimulated for 120 min with the different M proteins (2.5 μg/mL). The samples were centrifuged at 2,000 × g for 10 min, and the CD62P, CD40L, and platelet factor 4 content in the supernatants was measured using commercially available ELISA kits (R&D Systems) according to the manufacturer’s instructions. HEPES buffer alone was used to determine the background levels of CD62P, CD40L, and platelet factor 4, and the platelet agonist thrombin (1 U/mL; Triolab) was used as a positive control for platelet activation in combination with the anticoagulant peptide Gly-Pro-Arg-Pro (1.25 mg/mL; Bachem).
3
Flow Cytometric Assessment of Platelet Activation
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The flow cytometric assessment of platelets was performed using FACSCalibur (Becton Dickinson, Heidelberg, Germany) [12 (link)]. Diluted PRP aliquots (5 × 107/mL) were fixed and stained with FITC-labeled monoclonal surface antibody against glycoproteins (GP) CD41 (GPIIb/IIIa-complex, integrin αIIbβ3), CD42a (GPIX) and CD42b (GPIb) (Coulter, Immunotech, Marseille, France). FITC-labeled anti-vWF (Bio-Rad AbD Serorech, Puchheim, Germany) and Alexa Fluor 488-labeled anti-fibrinogen (Invitrogen, Waltham, MA USA) were used to stain the platelets. In the presence of 1.25 mM Gly-Pro-Arg-Pro (Bachem, Bubendorf, Switzerland) diluted PRP (5 × 107 platelets/mL) was stimulated with different concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5, and 1 U/mL; Siemens Healthineers, Marburg, Germany) to conduct the CD62 and CD63 expression analyses. Additionally, the platelets were stained with monoclonal FITC-labeled anti-CD62 (P-selectin) and anti-CD63 antibodies (lysosomal membrane associated glycoprotein 3, Immunotech, Marseille, France). Data of patients and controls (day control and 20 independent measurements from 10 controls as mean ± standard error of the mean, SEM) were analyzed using GraphPad Prism software (version 8, San Diego, CA, USA).
4
Investigating Platelet Activation by M1 Protein
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Flow cytometry was used to investigate platelet activation in response to M1 protein as previously described [21 (link)]. PRP from five healthy donors was diluted 2:3 in 1 mm HEPES buffer pH 7.4 and stimulated with M1 protein (2.5 μg/mL) for 15 min at room temperature. HEPES buffer alone was used to determine the background platelet activation, and the platelet agonist thrombin (1 U/mL, Triolab) was used as a positive control for platelet activation during hemostasis. The anticoagulant peptide Gly-Pro-Arg-Pro (1.25 mg/mL; Bachem) was added prior to stimulation with thrombin to prevent fibrin polymerization and coagulation. After stimulation, the samples were incubated with anti-CD62P-PE (1:10) (BD Biosciences, clone AC1.2) for 15 min at room temperature protected from light. Platelet counts were acquired on an Accuri C6 Plus flow cytometer (BD Biosciences), and the data were analyzed using C6 Plus Software. Platelets were gated based on size and granularity in logarithmic mode, and the CD62P intensity for the gated population was analyzed in histograms.
5
M Protein-Induced Platelet Activation Assay
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Flow cytometry was used to investigate platelet activation in response to M proteins as previously described (25 (link)– (link)27 (link)). PRP was prepared from 10 healthy donors (donor 1–10) and diluted 2:3 in 1 mM HEPES buffer, pH 7.4, and stimulated for 10 min with the different M proteins (2.5 μg/mL). HEPES buffer alone was used to determine the background platelet activation, and the platelet agonist thrombin (1 U/mL; Triolab) was used as a positive control for platelet activation in combination with the anticoagulant peptide Gly-Pro-Arg-Pro (1.25 mg/mL; Bachem). After stimulation, the samples were incubated with anti-CD62P-PE (1:10) (BD Biosciences; clone AC1.2), to detect activated platelets, for 15 min at room temperature and protected from light. The samples were run on an Accuri C6 Plus flow cytometer (BD Biosciences), and the data were analyzed using C6 Plus software. Platelets were gated based on size and granularity in logarithmic mode, and the CD62P intensity for the gated population was analyzed in histograms (Fig. S2). In order to investigate the role of IgG for M protein-mediated platelet activation, the PRP was pretreated with IdeS as described above for ELISA.
6
Platelet flow cytometry analysis
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Flow cytometry analyses were performed using FACSCalibur (Becton Dickinson, Heidelberg, Germany) (Lahav et al., 2002 (link)). Diluted PRP aliquots (5 × 107 platelets/ml) were fixed and stained with FITC-labeled monoclonal surface antibody against CD41 (GPIIb/IIIa-complex), CD42a (GPIb/IX) and CD42b (GPIb) (Coulter, Immunotech, Marseille, France). FITC-labeled anti-VWF (Bio-Rad AbD Serorech, Puchheim, Germany) and Alexa Fluor 488-labeled anti-fibrinogen (Invitrogen, Waltham, MA United States) was used to stain the platelets. In the presence of 1.25 mM Gly-Pro-Arg-Pro (Bachem, Bubendorf, Switzerland) diluted PRP (5 × 107 platelets/ml) was stimulated with a number of concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5, and 1 U/ml; Siemens Healthineers, Marburg, Germany) to conduct the CD62 and CD63 expression analyses. Additionally, the platelets were stained with monoclonal FITC-labeled anti-CD62 (P-selectin) and anti-CD63 antibodies (lysosomal membrane-associated glycoprotein 3, LAMP-3; Immunotech, Marseille, France). Data of patients and controls (day control and 20 independent measurements from 10 controls as mean ± standard error of the mean, SEM) were analyzed using GraphPad Prism software (version 8, San Diego, CA, United States).
7
Platelet Flow Cytometry Analysis
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Flow cytometry analyses were performed according to Lahav et al. (Lahav et al., 2002 ) using FACSCalibur (Becton Dickinson, Heidelberg, Germany). Aliquots of diluted PRP (5 × 107 platelets/mL) were fixed and stained with FITC‐labeled monoclonal surface antibody against CD41 (fibrinogen receptor GPIIb/IIIa‐complex), CD42a (von Willebrand factor [VWF] receptor GPIb/IX), and CD42b (VWF receptor GPIb) (Coulter, Immunotech, Marseille, France), respectively.
For VWF‐binding analyses, diluted PRP (5 × 107 platelets/mL) was stimulated with different concentrations of ristocetin (0–1 mg/mL) and ADP (0–2 μmoL/L) for 3 min at RT, respectively. Platelets were stained with FITC‐labeled anti‐VWF (Bio‐Rad AbD Serotec, Puchheim, Germany) and Alexa Fluor 488‐labeled anti‐fibrinogen (Invitrogen, Waltham, MA, USA).
For secretion analyses, diluted PRP (5 × 107 platelets/mL) was stimulated with different concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5, and 1 U/mL; Siemens Healthineers, Marburg, Germany) in the presence of 1.25 mM Gly‐Pro‐Arg‐Pro (Bachem, Bubendorf, Switzerland). Platelets were stained with monoclonal FITC‐labeled anti‐CD63 antibody (lysosomal membrane associated glycoprotein 3, LAMP‐3; Immunotech, Marseille, France).
For VWF‐binding analyses, diluted PRP (5 × 107 platelets/mL) was stimulated with different concentrations of ristocetin (0–1 mg/mL) and ADP (0–2 μmoL/L) for 3 min at RT, respectively. Platelets were stained with FITC‐labeled anti‐VWF (Bio‐Rad AbD Serotec, Puchheim, Germany) and Alexa Fluor 488‐labeled anti‐fibrinogen (Invitrogen, Waltham, MA, USA).
For secretion analyses, diluted PRP (5 × 107 platelets/mL) was stimulated with different concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5, and 1 U/mL; Siemens Healthineers, Marburg, Germany) in the presence of 1.25 mM Gly‐Pro‐Arg‐Pro (Bachem, Bubendorf, Switzerland). Platelets were stained with monoclonal FITC‐labeled anti‐CD63 antibody (lysosomal membrane associated glycoprotein 3, LAMP‐3; Immunotech, Marseille, France).
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