Edta coated collection tubes

Whole blood was drawn from athymic nude mice by cardiac puncture immediately following CO₂ asphyxiation and collected in serum separator tubes (BD Biosciences) or EDTA coated collection tubes (BD Biosciences). 25 μL of whole blood was collected in EDTA coated tubes and analyzed by the Research Resource Center (RRC) facility at the University of Louisville for complete blood count (CBC) analysis. After 1 hour post-collection, whole blood samples collected in serum separator tubes were centrifuged for 10 minutes at 10,000 g. 250 μL of serum was removed from each sample, collected in a 1.5 mL eppendorf tube, and analyzed by the RRC facility for non-steroidal anti-inflammatory drug (NSAID) toxicological analysis. Liver damage was assessed by levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALKP). Kidney damage was assessed by levels of blood urea nitrogen (BUN) and creatinine (CREA).

Methamphamphetamine Hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Mice were weight-matched and randomly divided into groups and were administered a gradual escalating METH dose from 0.45–10 mg/kg over 6 days, followed by a single dose of 10 mg/kg/day subcutaneously (s.c. at the nape of the neck) until the mice were sacrificed. Once daily injection of METH was established, mice were intravenously injected with LCMV Clone 13 (2 × 10⁶ pfu). Experimental scheme is shown in Figure 1A. Peripheral blood was collected via submandibular vein into EDTA coated collection tubes (BD Biosciences) every 7 days until the end of the experiment. METH levels in plasma were assayed using a sensitive Elisa kit from Abnova (Taipei City, Taiwan). To determine the steady-state levels of METH upon daily injection, mice were bled at the same time at every time-point, to eliminate bias in the half-life of METH. Blood was collected after 2 h of METH injection at each time-point.

METH treatment and LCMV infection were performed as described [14 (link)]. Methamphamphetamine Hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). It has been shown that a good percentage of recreational METH abusers initially use lower doses and progressively increase the dose and eventually engage in increased amount and frequency of drug intake [18 (link)–20 (link)]. We used this rationale in this current study and to simulate a similar pattern, we used an escalating METH dose schedule. Mice were weight-matched and randomly divided into groups and were administered a gradual escalating METH dose from 0.45–10 mg/kg over 6 days, followed by a single dose of 10 mg/kg/day subcutaneously (s.c. at the nape of the neck) every day until the mice were sacrificed. Once a daily injection schedule of METH was established, mice were intravenously injected once with LCMV Clone 13 (2x10⁶ pfu). Peripheral blood was collected via submandibular vein into EDTA-coated collection tubes (BD Biosciences) every seven days until the end of the study. Mice were sacrificed at different time-points (days 14, 28 and 56) and spleens were collected and processed for flow-cytometric analysis. At least 3 mice were analyzed in each group for each time-point.