Pac 1 fitc

Flow cytometry reagents such as anti-CD61/PErCP, anti-CD61/PE, anti-CD61/FITC, anti-CD62/PE, PAC-1/FITC, and Cellfix were obtained from Becton Dickinson (San Diego, CA, USA). ADP and collagen were purchased in Chrono-Log (Havertown, PA, USA). All other chemicals were of analytical grade or highest quality available products. Human sP-selectin ELISA kit (Invitrogen).

SAA was purchased from Plant Bio-Engineering Co. Ltd. (Purity 98%, Xi’an, China). Flow cytometry reagents, such as anti-CD62p/PE and PAC-1/FITC, were obtained from Becton Dickinson (San Jose, CA, USA). ADP and thrombin were purchased from Helena Laboratories (Beaumont, TX, USA) and Xinfan Bio-technology Co, Ltd. (Hangzhou, China), respectively. All other chemicals were of analytical grade or products of the highest quality available.

Prior to perfusion, microfluidic devices were coated with 2% w/v BSA for 10 min prior to wash out with unmodified tyrodes buffer pH 7.4. Hirudin anticoagulated whole blood was either perfused through the external syringe pump line as a baseline control (no microfluidic perfusion) or θ_e = 80° geometry at Q = 200 μL/min, or no perfusion as a resting control. Each sample was diluted 1:10 in 1× Tyrode’s buffer (+1 mM CaCl₂ + 0.5 % BSAw/v) and 5 μL of the diluted sample was immediately transferred to their respective Eppendorf tubes containing α−CD42bAPC (Becton Dickinson), and either 5 μL PAC-1 FITC (Becton Dickinson) or 5 μL α−P-selectin-PE (Life Technologies) . The tube containing positive control was activated with 40 μM ADP and all samples were let to rest for 15 min. Reaction was stopped by adding 600 μL 1× Tyrode’s buffer and immediately transferred to 12 × 75 mm Falcon® polystyrene test tubes, and all samples were assessed within a 2-h window. Platelets were identified by light scatter characteristics and confirmed by CD42b expression. Positive controls were used to adjust voltages. Acquisition was performed on a BD FACSCanto-II (Becton Dickinson) that was routinely calibrated with Calibrite beads in conjunction with FACSComp Version 5.1 software (Becton Dickinson). The results were analyzed with FlowLogic 7.2.1 flow cytometric analysis software.

Adenosine receptor agonists were purchased from Sigma (St. Louis, MO, USA) (NECA (CAS № 35920-39-9)), and Cayman (Ann Arbor, MI, USA) (regadenoson (CAS № 313348-27-5)). LUF5835 (2-amino-6-(1H-imidazol-2-ylmethylsulfanyl)-4-(3-hydroxy-phenyl) pyridine-3,5dicarbonitrile) was synthesized at Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland. Cangrelor (AR-C69931MX) was from Cayman Chemical (Ann Arbor, MI, USA). Prasugrel metabolite (R-138727) was obtained from BOC Sciences (Shirley, NY, USA). Calcein AM was obtained from Molecular Probes (Eugene, OR, USA). Antibodies anti-human CD61/PErCP, CD61/PE, CD62/PE, PAC-1/FITC, mouse IgG1/PE isotype control, mouse IgG1/FITC isotype control, Cellfix, buffered sodium citrate was purchased from Becton-Dickinson (San Diego, CA, USA). Phosphate buffered saline pH 7.4 (PBS) was obtained from Corning (New York, NY, USA). Dimethyl sulfoxide (DMSO), adenosine diphosphate (ADP), and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). All other chemicals, unless otherwise stated, were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

Bactopeptone, bis-benzimide, glutaraldehyde, penicillin, propidium iodide, sodium citrate, streptomycin, trypsin, and yeast extract were acquired from Sigma Aldrich (Saint Luis, MO, USA); anti CD61-PerCP, anti CD62-PE, and PAC-1-FITC (Becton-Dickinson, Franklin Lakes, NJ, USA); Hoechst 33342 from Santa Cruz Biotechnology (Dallas, TX, USA); McCoy’s 5A medium and fetal bovine serum (FBS) from Biowest (Nuaillé, France) and XTT test from Biotium (Fremont, CA, USA). Bromophenol blue, dithiothreitol (DTT), glycerol, iodoacetamid, Immobiline Dry Strip gels, pH 4–7, Precast 12.5% polyacrylamide gel and buffer for 2-D DIGE, sodium dodecyl sulfate (SDS), Tris-HCl, UREA, (GE Healthcare, Little Chalfont, UK). Refraction-2D Labeling Kit and DyeAgnostics was obtained from LKB Biotech, Warsaw, Poland. All other reagents were obtained from POCH SA (Gliwice, Poland). Standard polystyrene flasks (T-75 flasks) and flat bottom cell culture microplates (12, 24, 48 and 96 wells) were from TPP Techno Plastic Products AG (Trasadingen, Switzerland). All other disposables were from VWR Int. (Gdansk, Poland).