Bio plex pro mouse cytokine standard 23 plex

The mice were deeply anesthetized using tribromoethanol (Sigma-Aldrich, St. Louis, MO, USA). Blood was taken by removal eyeball and centrifuged at 1000 × g/min for 10 min at 4 °C to obtain plasma for measurement cytokines using the Bio-Plex Pro Mouse Cytokine Standard 23-Plex (Bio-Rad) according to the manufacturer’s protocol. The cytokine such as tumour necrosis factor (TNF)-α, IL-1β, and IL-6 was secondly validated with the ELISAs reagents (Absin, Shanghai, SH, China) and performed according to the manufacturer’s instructions. Cytokine concentrations are expressed in pg/mL. The mice underwent cardiac perfusion with 0.01 M PBS (Sigma-Aldrich, St. Louis, MO, USA). The brain hemispheres were placed on an ice-cold glass dissection plate and orientated in the sagittal plane. The right hemispheres were placed in 4% paraformaldehyde for cryostat sectioning and immunohistochemistry and stored at − 80 °C until required.

Brain and plasma samples were collected at 6 h, 1 day, 3 days, 7 days and 14 days after HI. Briefly, mice were deeply anaesthetised, blood was collected from the heart’s right ventricle and transferred to EDTA-coated tubes and set aside for further processing, animals were then transcardially perfused with ice-cold 0.9% saline and brains were rapidly removed and frozen on dry ice. Plasma samples were isolated via centrifugation (10 min, 1000×g, 4 °C) and frozen on dry ice prior to storage at − 80 °C.
Brain lysates were prepared through mechanical dissociation of tissue samples in 600 μl of lysis buffer (10 mM EDTA, 1% Triton-X-100, 1% Protein Inhibitor Cocktail [Sigma-Aldrich#8340] in RNase-free PBS) followed by sonication and centrifugation (4500×g, 5 min, 4 °C). Protein concentration was assessed using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer’s protocol, and the final concentration of the samples was adjusted to 1 mg/ml using the lysis buffer. Preparations were carried out at 4 °C, and samples were stored at − 80 °C.
Cytokine concentration in brain lysates and plasma samples were assessed using a Bio-Plex Pro Mouse Cytokine Standard 23-Plex (Bio-Rad) kit in accordance with manufacturers’ instructions on a Bio-Plex 200 analyser. For brain samples, the results were normalised to the brain protein concentration.