Lithium heparin

In order to mimic the plasma situation, serum (from SST, 5B tubes) from the same 6 donors (above) was spiked with K 2 EDTA, Lithium heparin or sodium citrate. Lithium heparin and K 2 EDTA were obtained from Sigma (product nr H0878 and 03660, respectively) and sodium citrate buffer was collected from BD 3B tubes. K 2 EDTA concentration in the BD tubes 7B is present at 1.8 mg/mL whereas the BD 6B tubes contain 17 units Lithium heparin per ml blood. K 2 EDTA was prepared at 18 mg/L and 10 µL was evaporated to dryness under N 2 (g). By adding 100 µL serum 1 × K 2 EDTA was obtained (1.8 mg/mL serum). Lithium heparin was prepared at 170 Units/ml MQ water and 10 µL (1.7 Units) was evaporated under N 2 (g). Addition of 100 µL serum resulted in 1 × Lithium heparin. Also 10 × higher concentrations were tested for K 2 EDTA and Lithium heparin. Sodium citrate buffer (collected from the 3B tubes) was added to serum at the same standard ratio of 1:9 as applied in the 3B tubes preparing sodium citrate plasma. Maximal storage time for the spiking experiments was 12 months.

Fish were carefully removed from the aquaria, anaesthetised (0.5 g/L, neutralized MS222; Acros Organics, Geel, Belgium), quickly blotted dry and weighed followed by sampling. Blood was drawn from the caudal vessels using a heparinised needle and syringe within 1 min of sedation (2500 unit/ml lithium heparin, Sigma, Munich, Germany). The blood was immediately centrifuged to 5,000 g at 4 o C for 1 min. Plasma was separated into three 500 µl Eppendorf bullet tubes. Gills, kidney, liver and muscle tissues were wrapped in aluminium foil. All samples were immediately frozen in liquid N2 and stored at -80 o C for further analysis.

The spleen tissues were pressed through sterilized nylon mesh (40 µm, Dutscher)
with Leibovitz 15 (L15) medium (Sigma) containing heparin lithium (10 U.mL -1 , Sigma), penicillin (500 U.mL -1 , Biochrom AG) and streptomycin (500 µg.mL -1 , Biochrom AG) to obtain leucocyte suspension. After storage of 12 hours at 4 ± 1°C, L15 medium-diluted samples were loaded onto Ficoll gradient (Histopaque®1077, density of 1,077g.mL -1 , Sigma). After centrifugation (400 g, 30 min, 15 °C), leucocytes enriched suspensions were collected and washed twice in L15 medium (300 g, 5 min, 4 °C). Then, leucocytes were adjusted at 10 6 cells.mL -1 with Malassez haemocytometer to perform cytometer analyses.
Analyses were carried out on whole leucocytes, using a Cyan TM ADP flow cytometer (Beckman Coulter). For each leucocyte sample, 10,000 cells were counted.
Leucocyte differential was obtained using forward scatter (FSC) and size scatter (SSC) parameters for size and granularity, respectively. The cellular mortality percentage and the phagocytosis activity were determined, using propidium iodure (1.0 g.L -1 , Molecular Probes) and Fluorescent microsphere (2.7*10 10 particles.mL -1 , Fluorospheress carboxylatemodified microsphere, diameter 1 mm, Molecular Probes), respectively, according to Bado-Nilles et al. (2009a) .

DNA damage were assessed using comet assay described by (Santos et al. 2013) (link), with minor modifications, described below. Blood samples were kept at 4 °C in 800 µL of L15 medium Leibovitz (L15, Sigma) modified with heparin lithium (20 U/mL, Sigma), penicillin (500 U/mL, Sigma) and streptomycin (500 µg/mL, Sigma) until analysis (48 h after blood collection). Slides were transposed for 20 min instead of 40 min in an electrophoresis tank filled with electrophoresis buffer (300 mM NaOH, 1 mM EDTA, qsp 1.5L of osmotic water, Sigma for both products). Several genotoxicity parameters were recorded using Comet Assay 4 software. One hundred cells per slide (two slides per sample) were randomly selected for analysis.