Male crl cd1 icr mice

Male Crl:CD1 (ICR) mice (Charles River Canada Inc.) were injected intravenously (IV) with a single dose of AD-114-Im7-FH at 3 mg/kg (3 mL/kg) and blood samples were collected 5, 15, 45, 60 and 90 min post dosing. AD-114-Im7-FH was quantified in plasma by LC-MS/MS (Algorithme Pharma, Canada) by monitoring the signature peptide LTPNQQR. Twenty-five μL of thawed plasma was combined with 25 μL of 8 M urea and incubated at 60 °C for 30 min. Twenty-five μL of stable labelled (¹³C¹⁵N) internal standard (IS) peptide GEKLTPNQQR*IG (0. 25 μg/mL) was added, followed by 200 μL of trypsin (2 mg/mL in 100 mM NH₄HCO₃). The sample was vortexed briefly then incubated at 60 °C for 90 min. Fifty μL of 10% HCOOH was added followed by vortexing and then centrifugation at 14 000 rpm for 2 min, 175 μL of supernatant was then transferred to a clean tube ahead of LC-MS/MS using a AB Sciex QTRAP5500 triple quadrupole mass spectrometer. Reverse phase chromatography was performed over a XBridge BEH300 C18, 50 × 2.1 mm, 3.5 μm column at 60 °C with a flow rate of 600 μL/min using mobile phases A [0.1% (v/v) aqueous HCOOH] and B [0.1% (v/v) HCOOH in acetonitrile].
The MS instrument used a turbo spray ESI ion source in positive mode and the multiple reaction monitoring (MRM) as method.

Male Crl:CD1 (ICR) mice (approximately 25–30 g, 3–4 months old) were purchased from Charles Rivers Laboratories (Barcelona, Spain). They were housed in cages maintained in standard conditions (Centro de Experimentación y Conducta Animal, Universidad de Málaga) at 20 ± 2 °C room temperature, 40 ± 5% relative humidity, and a 12h light⁄dark cycle with a dawn/dusk effect. Water and standard rodent chow (Prolab, Saint Louis, MO, USA; RMH 2500, 2.9 kcal/g) were available ad libitum.
The animals were handled daily for 10 min and habituated to an oral gavage procedure for 1 week before experimentation in order to minimize stress effects.

Male Crl:CD1 (ICR) mice (Charles River Canada Inc.) were injected IV with AD-114-PA600-6H at 10 mg/kg (3.1 mL/kg) and blood samples were collected at 15, 30 min, 1, 2, 3, 4, 8, 12, 18, 24, 36, 48 and 72 h post dosing. Plasma samples were diluted 1/10 in 1% (w/v) BSA (Sigma) in PBS and AD-114-PA600-6H was quantified by ELISA using anti-human NCAM-1 (R&D Systems) to capture i-body and anti-Histidine-HRP (BioRad) to detect His₆ tagged i-body. Absorbance of the HRP reactive reagent 3,3´,5,5´-tetramethylbenzidine (TMB) was quantified at 450 nm using a Biotek Powerwave XS or Synergy HT microplate reader (Biotek).