Z2 coulter particle counter

Clot retraction was performed in whole-blood and platelet-rich plasma (PRP). Whole-blood clot retraction was induced in the absence of anticoagulants according to a standard clinical method [28 (link)]. Briefly, blood (500 μL) was transferred to a 10 × 75 mm disposable glass tube and allowed to stand still for 2 h at 37 °C. The clot was then removed, and the amount of serum was measured to determine the percentage of clot retraction.
Clot retraction in PRP was measured in the presence of an anticoagulant by a method modified by Owens et al. [29 (link)]. Whole blood anticoagulated with citrate, PPACK, or heparin was centrifuged at 150× g for 15 min at 27 °C to obtain PRP. Platelet counts were then determined by a Coulter Z2 particle counter (Beckman Coulter, Miami, FL, USA) and diluted to 2–2.5 × 10⁸ platelets/mL using autologous platelet-poor plasma (PPP) obtained by centrifugation of PRP at 900× g for 10 min. PRP was mixed with 100 U/mL of human α-thrombin (kindly provided by Dr. JW Fenton of the New York State Department of Health) in a total volume of 600 μL and transferred to a 22 × 72 mm disposable glass tube. After incubation for 2 hrs at 37 °C, the clot was removed, and the amount of serum was measured.

Mice were euthanized by sodium pentobarbital (Euthasol™ Schering-Plough, Lot# 1JRR11V), and the lungs with the heart were removed. Lung lavage was performed using ice-cold PBS (pH 7.4). Lung lavage cells were isolated by centrifugation (400× g, 5 min, 4 °C) and cell counts obtained using a Coulter Z2 particle counter (Beckman Coulter, Miami, FL, USA).

The SpPdp11 strain was grown in tryptone soya broth (OXOID Ltd., Basingstoke, UK) supplemented with 1.5% NaCl (TSBs, 20 h, 20 °C) under continuous shaking. Contant shaking was used to wash the bacteria that were removed from the plates in sterile phosphate-buffered saline (PBS, pH 7.4). The density of the bacterial suspension was resolved utilizing Coulter Z2 particle counter (BECKMAN Coulter, Barcelona, Spain), and the volume was adjusted to the target concentration (10⁹ cfu g⁻¹). This dose was chosen based on the health benefits in S. aurata and S. senegalensis as reported in previous studies [27 (link),29 (link)]

Approximately 10,000 cells per well (for SK-BR-3 and SK250LR) or 20,000 cells per well (for BT-474 and BT250LR) were seeded in triplicate on 24-well tissue culture plates, allowed to adhere and enter log phrase overnight. Each triplicate was treated with the indicated agents for 3 days. Culture medium was discarded. Cells were washed with PBSA twice and then detached from the well with 500 μl of 0.25% trypsin-EDTA solution. Trypsinised cells were diluted using Coulter Isoton II diluent and counted using a Coulter Z2 particle counter (Beckman Coulter); only the cell population with the size of 13–27 μm was counted.