Citrate phosphate dextrose solution

Male BALB/c mice, between 3 to 6 months of age (Charles River Laboratories, Inc., Boston, MA) and adult female C3H/HeN (Harlan, Inc) non-pregnant mice, were used for adult mouse studies. Spleens from fetuses of gestational age 18 days were isolated from pregnant female C3H/HeN mice. The use of animals was approved by the Institutional Animal Care Use Committee at Loma Linda University. BM cells were flushed from the femurs with 1 ml of sterile PBS (Cellgro, Manassas, VA). PB from BALB/c mice was obtained from the hepatic portal vein. A citrate-phosphate-dextrose solution (Sigma-Aldrich, St. Louis, MO) anticoagulant was added to blood in a 1.4 to 10 ratio. PBMCs were isolated using RBC lysis buffer method. Splenocytes from BALB/c mice were isolated by straining spleens through a 70 μM cell strainer (BD Falcon, Franklin Lakes, NJ) to create a single cell suspension in PBS. Fetal mouse spleens were processed using the same method as adult spleens.

Peripheral blood samples from adults were obtained from healthy volunteers at the National Institutes of Health (NIH) after informed consent with approval from the International Review Board (IRB) of the NIH (NIAID Protocol number 03-I-0263) and provided de-identified. Non-identifiable umbilical cord blood samples were obtained with the approval of the IRB of the Walter Reed National Military Medical Center (WRNMMC) (Protocol number WRNMMC-EDO-2018–0203). Umbilical cord blood was collected after delivery of the placenta into citrate-phosphate-dextrose solution (Sigma). Human blood was diluted in an equal volume of PBS and layered over Ficoll-Paque PREMIUM (GE Healthcare, Silver Spring, MD). MCs were isolated by centrifuging at 400×g for 30 minutes. MCs were washed twice and frozen in FCS/10% DMSO. On day of stimulation, MCs were thawed, washed, and rested in 48-well plates for 2 hours. Human IFNβ (PBL Assay Science) or R848 (InvivoGen, San Diego, CA), was then added for stimulation at 37°C for 16–18 hours prior to analysis by flow cytometry.

Murine RBCs were isolated from whole blood and transfused as previously described [12 (link)]. Briefly, whole blood was drawn from WT mice via intracardiac puncture and collected in sterile syringes. Citrate-phosphate-dextrose solution (Sigma-Aldrich, St. Louis, MO, USA) was added to the whole blood at a ratio of 1:9. The whole blood was leukoreduced with leukoreduction filters (Purecell Neo; Pall Medical, Port Washington, NY, USA). The filters were washed with sterile PBS, and the eluent was centrifuged at 800 g for 10 min. RBCs were combined with storage solution (150 mmol/l sodium chloride, 45 mmol/l dextrose, 29 mmol/l mannitol, and 2 mmol/l adenine, 3:1 ratio of RBCs to storage solution) and transferred to 100ml plasticizer storage bags (Charter Medical, Winston-Salem, NC, USA). The bags were heat sealed and stored at 4°C for 12–14 days before transfusion. Stored blood was warmed to room temperature, washed and concentrated to 60–70% haematocrit prior to transfusion at 10 μl/g into recipient mice through tail-vein injection. The animals were sacrificed 6 h following transfusion with intraperitoneal ketamine/xylazine (80/10 mg/kg) and plasma was obtained.