Acid citrate dextrose solution

Experiments involving animals were approved under Stanford APLAC protocol number 31511. Seven-week-old female BALB/cJ mice ( Jackson Laboratory) were injected with PBS or 10 mg per kg (body weight) αHER2-eStcE via i.p. injection every other day (days 1, 3, 5 and 7) for a total of four doses. On day 8, mice were fasted for 4 h in cages without food or bedding. After fasting, blood was collected via tail vein nick, and 15% (vol/vol) acid citrate dextrose solution (Sigma) was added. Mice were given an oral gavage (150 μl) of 80 mg ml⁻¹ FITC–dextran (4 kDa) dissolved in PBS. After 4 h, blood collection was repeated. All blood samples were centrifuged at 5,000 r.p.m. for 10 min to isolate plasma. Plasma samples were diluted 1:10 in 100 μl of PBS and transferred to a black, opaque-bottom 96-well plate. A serial dilution of FITC–dextran (0.2 to 12.5 μg ml⁻¹ range) in PBS with 10% (vol/vol) mouse plasma was included for comparison. Fluorescence signal (excitation: 485 nm; emission: 540 nm) was measured using a SpectraMax i3x microplate reader.

564Igi mice were genotyped using digital droplet PCR (ddPCR). Tail DNA was isolated and digested with AluI (NEB). Droplets were prepared from a mix of tail DNA, primers (Supplementary Table 2), and EvaGreen Supermix (Bio-Rad) using a QX200 Droplet Generator (Bio-Rad). PCR was performed using a C100 Touch Thermal Cycler (Bio-Rad) and droplets were read on a QX200 Droplet Reader (Bio-Rad). 564Igi heavy (Hi) and light (Ki) chain copy number was quantified by comparing to amplification of reference mRPP30 using QuantaSoft (Bio-Rad). FACStyping of CD45.1 mice and bone marrow chimeras was performed by bleeding mice retroorbitally using heparinized capillary tubes and collecting into 30 μL of acid-citrate-dextrose solution (Sigma). Stabilized blood was underlayered with 1 mL of Lymphocyte Separation Medium (Corning) and centrifuged at 400 × g for 30 min at room temperature. The mononuclear cell layer was aspirated and processed for flow cytometry as described below using anti-CD45.1 and anti-CD45.2 antibodies.

Chicken feathers were gotten from the local market. Polyacrylonitrile (PAN) with MWT of 150,000 g/mol was purchased from Sigma Aldrich (USA). N,N-Dimethyl Formamide (C₃H₇NO) 99.0%, sodium hydroxide (NaOH), formaldehyde (36%), calcium chloride, PBS buffer (pH 7), and acetic acid (CH₃COOH) were purchased from El-Goumhouria Company, Alexandria, Egypt. Acid citrate dextrose solution (ACD) was purchased from Sigma-Aldrich (Chemie GmbH, Steinheim, Germany). Gram-negative Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa) and gram-positive Staphylococcus aureus ATCC 25923 (S. aureus) bacteria were investigated for antibacterial tests. For biomedical tests (hemolysis and thromogenicty tests), the blood samples were obtained from rabbits.

Whole blood citrate dextrose samples were centrifuged at 200 x g for 15 min at RT (with no brake applied). The top 3/4 of the platelet-rich plasma (PRP) was transferred into a new plastic tube, without disturbing the buffy coat layer, and was centrifuged as above for 5 min to remove by pelleting the residual erythrocytes/white blood cells. The 2/3 of supernatant was withdrawn and centrifuged in Acid-Citrate-Dextrose (ACD) solution (1-part ACD solution to 9 parts blood) (Sigma-Aldrich, S. Louis, MO, USA) at 800 x g for 20 min (with no brake applied). Platelet pellets were gently washed and resuspended in Hepes-Tyrode buffer pH 7.4 and aliquoted according to the final analyses. Before the flow cytometry analyses, to ensure that platelets cannot be induced to a new functional state, we used ThromboFix Platelet stabilizer (Beckman Coulter, Brea, CA); moreover, during all the procedures, strong mechanical forces (i.e., fast pipetting or vigorous shaking) have been avoided, while blood and reagents were always kept and handled at RT or 37°C.

Acid citrate-dextrose (ACD) solution was purchased from Sigma-Aldrich (St. Louis, MO). The following reagents of analytical grade or higher were obtained from Sigma-Aldrich: NAD+, NMN, 8-bromoadenosine-5’-monophosphate (BMP), bovine serum albumin (BSA), citric acid, trichloroacetic acid, ammonium acetate, ammonium formate, methanol, and acetonitrile. The chloride salt of NR was purchased from ChromaDex, Inc. (Irvine, CA; Catalog #: ASB-00014313-001), and d₄-NR triflate was purchased from Toronto Research Chemicals (Toronto, ON; Catalog #: N407772).

Cellular analyses were performed on platelets isolated from peripheral whole blood collected in citrate dextrose tubes as follows. Platelet rich plasma (PRP) was obtained by centrifugation of fresh blood at 200 x g for 10 min at room temperature (RT). PRP was transferred into a new plastic tube, without disturbing the buffy coat layer, and centrifuged as above for 5 min to remove the residual erythrocytes/white blood cells. PRP supernatant was withdrawn and centrifuged in Acid-Citrate-Dextrose (ACD) solution (1 part ACD solution to 9 parts blood) (Sigma-Aldrich) at 800 x g for 20 min at RT. Platelet pellets were gently washed and resuspended in Tyrode’s buffer (pH 7.8) and aliquoted according to the final analyses.
We performed a multicolour flow-cytometry for evaluating the surface protein expression of HYAL2 by staining isolated platelets with unconjugated HYAL2 antibody (ThermoFisher Scientific). The secondary antibody for the HYAL2 was Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000) (Invitrogen).We used CD45-ECD antibody (Beckman Coulter) for verifying the purity of our platelet samples and anti-CD62P-PE antibody (eBioscience, INC.) to determine platelet activation.