Sstii advance blood collection tubes

To assess in vivo intestinal permeability, mice were gavaged with FITC-dextran (44 mg/100 g body weight, 4 kDa, Sigma) 4 h prior to euthanasia (Gupta and Nebreda, 2014 (link)). Mice were anesthetized with a mixture of ketamine (90 mg/kg, Ketalar, Pfizer) and xylazine (10 mg/kg, Rompun, Bayer) (i.p.). Blood was collected via cardiac puncture and transferred into SSTII Advance Blood Collection Tubes (BD Vacutainer). After centrifugation (10,000 rpm, 5 min), serum was collected and diluted with an equal volume of PBS. Aliquots of 100 µl were transferred into a 96-well microplate, each sample in duplo. The concentration FITC in the serum was detected by fluorometry (Fluoroskan Microplate Fluorometer, Thermo Fisher Scientific) with an excitation wavelength of 480 nm and an emission wavelength of 530 nm. The exact FITC concentration in each well was calculated using a serially diluted FITC-dextran solution as a standard curve (0–8,000 ng/ml).

Blood was collected from mice by cardiac puncture and put into SSTII Advance Blood Collection Tubes (BD Vacutainer). After centrifugation (3500 rpm, 20 min), serum was collected and stored at −80°C for CBA.
The levels of IL-2, IL-4, IL-6, IFN-γ, TNF, IL-17A and IL-10 were measured in the serum samples using a CBA Mouse Th1/Th2/Th17 Cytokine Kit according to the manufacturer’s instructions (BD Biosciences). Blanco, standard and unknown samples were measured in duplo. The minimum detectable level was: 0.1 pg/ml for IL-2, 0.03 pg/ml for IL-4, 1.4 pg/ml for IL-6, 0.5 pg/ml for IFN-γ, 0.9 pg/ml for TNF, 0.8 pg/ml for IL-17A and 16.8 pg/ml for IL-10. The intra-assay variability (%CV) was 3% for IL-2, 3% for IL-4, 6% for IL-6, 3% for IFN-γ, 4% for TNF, 2% for IL-17A and 11% for IL-10. The inter-assay variability (%CV) was 3% for IL-2, 6% for IL-4, 4% for IL-6, 3% for IFN-γ, 5% for TNF, 4% for IL-17A and 6% for IL-10.