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Platelet Migration and Bacterial Trapping Assay under Flow.

Bio-protocol (2018-09-20)
Shuxia Fan, Michael Lorenz, Steffen Massberg, Florian Gaertner
ABSTRACT

Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Thrombin from bovine plasma, lyophilized powder, 40-500 NIH units/mg protein (biuret)
Sigma-Aldrich
Albumin human, Cellastim S, recombinant, expressed in rice, lyophilized powder, suitable for cell culture, low endotoxin, ≥96% (SDS-PAGE)
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
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Adenosine 5′-diphosphate sodium salt, bacterial, ≥95% (HPLC)
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Sodium citrate tribasic dihydrate, ACS reagent, ≥99.0%
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Ampicillin sodium salt
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HEPES solution, 1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture
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IPTG, ≥99% (TLC), ≤0.1% Dioxane
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Fibrinogen from human plasma, 50-70% protein (≥80% of protein is clottable)
Sigma-Aldrich
Dimethyl sulfoxide, sterile-filtered, BioPerformance Certified, meets EP, USP testing specifications, suitable for hybridoma