Alsever s solution

Whole blood was collected by retro-orbital bleeding using heparinized tubes from mice and mixed with an equal volume of Alsever’s solution (Sigma Aldrich, cat. number A3551). RBCs were opsonized with the mouse red blood cell antibody (Rockland, cat. number 210–4139); 20 μl of antibody was used for approximately 10⁹ RBCs.
For in vitro EP experiments, RBCs were added at a ratio of 1:10 (macrophage:RBCs) into cell culture medium and applied to BMDMs. One h later, medium was aspirated and cells were washed with DPBS and RBC lysis buffer (150 mM NH₄Cl, 10 mM NaHCO₃, 1.3 mM EDTA) before fresh medium was replaced.

Bacterial hemolytic activity was assessed using the microplate hemolysis assay described by Högfors-Rönnholm and Wiklund [51 (link)]. Blood was collected in an equal volume of Alsever’s solution (Sigma Aldrich) by caudal venipuncture of rainbow trout (about 800 g). Erythrocytes were centrifuged (1000 x g, 5 min, 4°C), washed three times with phosphate buffered saline (PBS, pH 7.2). For the assay, washed and packed erythrocytes were suspended to 5% (v/v) in PBS. An equal amount (30 μl) of erythrocyte and bacterial suspensions (including the phage resistant strain V1-20 as a negative control [13 ]) were mixed in triplicates into a U-well microtiter plate (Greiner bio-one) and incubated for 24 h at 10°C and 400 rpm rotation. Following incubation, 150 μl 0.5% NaCl was added to the wells and the plate was centrifuged (1000 x g, 5 min, 4°C). The supernatants were transferred to a F-well microtiter plate (Greiner Bio-one) and the absorbance was measured at 540 nm (A). A negative control (background, A^background) with only 0.5% NaCl and erythrocytes and a positive control (total hemolysis, A^100%) with distilled water and erythrocytes were included in triplicates on each plate. The hemolytic activity was calculated according to: Hemolytic activity = (A-A^background)/ (A^100%-A^background). Experiments were repeated in four independent assays.

T. lestoquardi-infected cell lines were obtained from peripheral blood or lymph nodes of infected sheep. Blood or lymph node aspirates taken from the draining lymph node nearest the site of challenge were collected daily in Alsever’s solution (Sigma Aldrich), from day 12 post-infection but before treatment with buparvaquone [35 (link)]. PBMC and lymph node mononuclear cells were separated from the blood or lymph node aspirate using Ficoll-Paque (GE Life Sciences) according to the manufacturer’s recommendations, and resuspended in culture medium (RPMI 1640 Glutamax medium, Life Technologies, Gibco, Paisley, UK) supplemented with 10% FCS (GIBCO), penicillin-streptomycin (5000 units ml^-1 and 5 mg ml^-1, respectively, Sigma-Aldrich, Dorset, UK) and 50 μM of 2-mercaptoethanol (Sigma Aldrich). PBMC and/or lymph node cells were counted and dispensed at 2.5 × 106–1 × 10⁷ cells per well in 2 ml culture medium in 24 well plates and cultured until parasitized cell lines were established [35 (link)]. The cultures were incubated at 37°C in 5% CO₂.

All reagents were of analytical grade and used as received without any purification: Ferric chloride hexahydrate (FeCl₃.6H₂O, 97%), ferrous chloride tetrahydrate (FeCl₂.4H₂O, 99%), ammonium hydroxide (NH₄OH_(ag), 28–30%), tetraethylorthosilicate (TEOS, 98%), vinyltrimethoxysilane (VTMS, 98%), toluene (99.9%), magnesium sulfate (MgSO₄, 99.5%), N-vinylcaprolactam (NVCL, 98%), methacrylic acid (MAA, 99%), ammonium persulfate (APS, 98%), N, N’-Methylenebisacrylamide (NMBS, 99%), Doxorubicin hydrochloride (DOX, 98–102%) Alsever´s solution, and phosphate buffered saline solution (PBS, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (99%) and isopropyl alcohol (99%) were purchased from JT Baker (Phillipsburg, NJ, USA).

ECFCs were obtained from human umbilical cord blood with appropriate maternal consent and under ethical approval in accordance with the Declaration of Helsinki. The mononuclear cell fraction was isolated by density gradient fractionation. Umbilical cord blood was diluted using Alsever's solution (Sigma-Aldrich) and carefully layered on Histopaque-1077 (Sigma-Aldrich) in a 1:1 ratio. The fraction at the interphase between the Histopaque and the plasma was carefully removed using a transfer pipette and resuspended in EGM-2 (Lonza Ltd.) supplemented with 20% fetal bovine serum (Hyclone), and plated in 24-well NUNC tissue culture plates precoated with rat tail collagen type I (BD Biosciences). ECFCs were characterized by immunophenotyping for CD31, CD105, CD14, and CD45 (eBioscience) using an Acoustic Focusing Cytometer (Attune NxT, Life Technologies) following already established methodology (5 (link)). For all experiments, ECFCs at passage 9 were used.