Mouse platelet aggregation after stimulated with thrombin (0.03 U/ml) or CRP (0.1 μg/ml) was detected in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, USA) at 37 °C with stirring (1000 rpm). ATP release was monitored in parallel with platelet aggregation using luciferin/luciferase reagent (Chrono-log Corporation). Platelet α-granule release as presented by P-selectin expression was measured by flow cytometry using PE-conjugated anti-P-selectin antibody as described previously [26 (link)]. The platelet αIIbβ3 integrin expression was measured using FITC-conjugated mouse anti-human CD41a antibody (αIIb) by flow cytometry.
Lumi aggregometer model 700
Manufactured by Chrono-Log
Sourced in United States
The Lumi-Aggregometer Model 700 is a laboratory instrument used for measuring the aggregation of platelets in blood samples. It measures the optical density changes that occur during platelet aggregation in response to various stimuli.
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10 protocols using lumi aggregometer model 700
1
Mouse Platelet Activation Assay
2
Platelet Aggregation Assay with C5a
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Aggregation of platelets was estimated with light transmission aggregometry using a lumiaggregometer Model 700 (ChronoLog). Washed murine platelets were adjusted to a concentration of 250 × 103 platelets per µl in Tyrode buffer pH 7.4 and were preincubated with PBS or 20 ng/ml C5a for 10 min. After adjusting the measurement according to the manufacturer’s protocol, platelets were activated with 0.25 µg/ml CRP or with 2.5 mU/ml thrombin for 10 min at 37 °C and a stirring speed of 1000 rpm. Analysis was performed using the aggrolink8 software (ChronoLog).
3
Platelet Aggregation Assay with C5a
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Aggregation of platelets was estimated with light transmission aggregometry using a lumiaggregometer Model 700 (ChronoLog). Washed murine platelets were adjusted to a concentration of 250 × 103 platelets per µl in Tyrode buffer pH 7.4 and were preincubated with PBS or 20 ng/ml C5a for 10 min. After adjusting the measurement according to the manufacturer’s protocol, platelets were activated with 0.25 µg/ml CRP, with 2.5 mU/ml thrombin, or further agonists for 10 min at 37 °C and a stirring speed of 1000 rpm. Analysis was performed using the aggrolink8 software (ChronoLog). For human samples, platelet-rich plasma (PRP) was generated as described above and used for aggregometry with platelet-poor plasma as a control; stimulation was performed using ADP and CRP as well as C5a at defined concentrations.
4
Platelet Aggregation and ATP Release Assay
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Platelet aggregation was conducted in the presence of fibrinogen (0.5 mg/ml) as described previously (Luo et al., 2019 (link); Wei G. et al., 2020 (link)). After matrine treatment, platelet aggregation induced by CRP (0.1 μg/ml) or thrombin (0.04 U/ml) was analyzed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, United States) at 37°C with stirring (1,000 rpm). Platelet aggregation was presented as a percentage of maximum platelet aggregation. ATP release was monitored in parallel with platelet aggregation after addition of luciferin/luciferase reagent (Chrono-log Corporation) according to the manufacturer’s instructions and quantified relative to the vehicle (0 mg/ml matrine) treatment.
5
Salidroside Modulates Platelet Aggregation
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After salidroside treatment, human platelet aggregation in response to thrombin (0.03 U/ml) or CRP (0.1 μg/ml) was assessed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, USA) at 37°C with stirring (1000 rpm). ATP release was monitored in parallel with platelet aggregation after addition of luciferin/luciferase reagent (Chrono-log Corporation) in accordance with the manufacturer's instructions. ATP release was presented as a percentage relative to the vehicle (0 μM salidroside) treatment. For mouse platelet aggregation, thrombin (0.02 U/ml) or CRP (0.05 μg/ml) was used.
6
Platelet Aggregation and ATP Secretion
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Platelet aggregation was performed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, USA) at 37 °C with stirring (1000 rpm) before and after stimulation. The ATP secretion was monitored in parallel with platelet aggregation after adding luciferin/luciferase reagent (Chrono-log Corporation) to platelet suspensions. ATP secretion was quantified relative to control mouse platelets.
7
Platelet ATP Secretion Quantification
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The ATP released from platelets (0.4 ml PRP adjusted to 3.0 × 108/ml) was measured by adding 50 μl of luciferin/luciferase reagent (CHRONO-LUME), 1 min before stimulation with 10 μM ADP. Platelet aggregation and ATP secretion responses were simultaneously measured, at 37 °C with stirring at 1000 rpm, in a Lumi-aggregometer Model 700 (Chrono-Log Co.). The amount of ATP (nmols) was determined using ATP standard calibration.
8
Platelet Aggregation Inhibition Assay
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Human venous blood was collected from healthy donors in compliance with the Declaration of Helsinki, which was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. All participants gave written informed consent for this study. Platelet-rich plasma (PRP) was obtained from the whole blood supernatants by centrifugation at 100 x g for 10 min. The cells were harvested, washed, and resuspended in phosphate-buffered saline (PBS; 1 × 107 cells/mL). For the antibody inhibition assay, cells were incubated with different concentrations of SZ163, SZ168, or control mouse IgG for 15 min on ice, followed by addition of 250 μL PRP. Platelet aggregation was measured in a Lumi-Aggregometer Model 700 (Chrono-log, Havertown, PA, USA). Data are provided as means ± SD of three independent experiments.
9
Platelet Aggregation and ATP Release
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Platelet aggregation was performed in the presence of fibrinogen (0.5 mg/ml). After ATRA treatment, platelet aggregation in response to collagen (5 g/ml) and thrombin (0.05 U/ml) was evaluated in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, USA) at 37°C with stirring (1000 rpm). Platelet aggregation was quantified as the percentage of maximum platelet aggregation in the absence of drug. The release of adenosine triphosphate (ATP) was monitored in parallel with platelet aggregation after addition of luciferin/luciferase reagent (Chrono-log Corporation) to the platelet suspension according to the manufacturer's instructions. ATP release was quantified relative to the vehicle (0 M ATRA) treatment.
10
Platelet Aggregation and ATP Release Assay
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Platelet aggregation and ATP release were carried out by optical aggregometry as described previously (19 (link)), using a lumi-aggregometer model 700 (Chrono-log Corporation), under continuous stirring at 1,200 rpm. Washed platelets were treated with ALR-S (20, 60 and 200 µg/ml) or a vehicle at 37˚C for 10 min. Luciferin/luciferase (10 µl) were added, and then stimulation was performed with collagen (1 µg/ml), thrombin (0.025 U/ml) or ADP (10 µM), respectively. Traces for aggregation and ATP release were recorded for 10 min at 37˚C. For H2O2 enhanced platelet aggregation, washed platelets were treated with ALR-S or a vehicle at 37˚C for 10 min, then the agonists were immediately joined into platelets after H2O2 (20 µM) was added.
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