Edta collection tube

Blood samples were obtained in the morning by venipuncture of the median cubital vein after overnight fasting, using commercial ethylenediaminetetraacetic acid (EDTA) collection tubes (BD Medical, Franklin Lakes, NJ, USA). Samples were immediately centrifuged at 1000× g for 10 min at 4 °C, aliquots were prepared, and stored at −80 °C until analysis.

Peripheral blood samples were obtained from volunteers by a licensed phlebotomist via venipuncture. Approximately 25 mL of blood per person was collected in EDTA collection tubes (BD Biosciences, Franklin Lakes, NJ) and labeled with a unique identifier. Heparinized blood was overlayed onto room temperature Ficoll (Sigma Aldrich, St. Louis, MO) in a 1:1 ratio in a sterile 50 mL conical tube. Samples were centrifuged at 400×g for 30 min at ambient lab temperature (~ 21 °C) with the brake off to ensure that deceleration did not disrupt the density gradient. Two milliliters of sera from each subject’s sample was stored at − 80 °C for subsequent antibody-reactivity testing. The buffy coat, containing the mononuclear cell layer, was extracted using a transfer pipette and washed three times with lipopolysaccharide (LPS)-free phosphate-buffered saline (PBS). Cells were resuspended in RPMI 1640 medium (Sigma, St Louis, MO) plus 10% human serum (50:50 male:female, Innovative Research, Novi, MI) and counted on a hemocytometer using a 1:1 Trypan blue (Sigma Aldrich, St. Louis, MO) exclusion (0.4% solution). Aliquots of 2 × 10⁸ viable cells/2 mL were cryopreserved using a method described previously [29 (link)] and infected at a later date.

After determining optimal timing for blood collection of in vivo LPS-activated neutrophils (n = 4), Dahl salt-sensitive hypertensive rats, genetically identical strain for hsICH rat model, (n = 2) were injected IV with 500µl LPS (Sigma Cat#L2630, 1.8 mg/kg in saline) 2hrs prior to blood collection. Five mLs of whole blood were collected in EDTA collection tube (BD Biosciences Cat#366450) under deep anesthesia, followed by euthanasia. The blood was brought to room temperature, layered onto a double gradient Histopaque (Sigma-Aldrich 10771 and 11191 respectively) as per manufacturer’s specifications (Millipore Sigma) and spun 800 x g for 30 minutes. Neutrophils were collected at the 1077/1191 interface, washed with 5 ml RPMI-1640 medium (Sigma Cat# R0883-500 ml) containing 1% BSA. In P96-well plates 50,000 live cells in 200 µL RPMI-1640 containing 1% BSA were incubated with 10a3 and 6g8 monoclonal antibodies (30 µg/ml) for 6 hours at 37°C and 5% CO2 respectively. Each condition was performed with 6 replicates. Live and dead cells were distinguished using trypan blue dye exclusion assay and counted in a phase contrast microscope.

Rats were injected intravenously with fresh LPS (Sigma Cat#L2630) 1.8 mg/kg in saline (final volume of 500 μl) 2 h prior to blood collection. Five mls of whole blood were collected in an EDTA collection tube (BD Biosciences Cat#366450) and diluted with 5 ml of Cell-based Assay Red Blood Cell lysis buffer (Cayman Cat#600611), mixed and layered over 3 ml of Histopaque (Sigma Cat#11191) in a 15 ml Falcon tube. The sample was spun 500 g for 30 min at RT. The yellow and clear top layer were discarded and the red pellet containing the RBCs and neutrophils were resuspended in 10 ml of Cell-based Assay Red Blood Cell lysis buffer (Cayman Cat#600611) and incubated for 10 min at RT to lyse the RBCs. Neutrophils were collected by centrifugation at 1200g × 10 min, washed with 5 ml RPMI-1640 medium (Sigma Cat# R0883) containing 1% BSA and resuspended in 2 ml RPMI-1640 containing 1% BSA. For the inhibition of survival assay, 50,000 live cells in 200 µl RPMI-1640 containing 1% BSA were seeded in P96 well plates. Each condition was performed in 4 replicates testing increasing concentrations of mAb (0.3–30 μg/ml). After 6 h at 37 °C, 5% CO₂ incubation, live and dead cells were counted by using trypan blue vital stain.