Cytokines, chemokines, and AnxA1 quantification

Dear Wei Li,

Thank you for your question.

Tissue homogenates were obtained following a previously described protocol (Amaral et al., 2011; Coelho et al., 2008; Vieira et al., 2017).

Briefly, tissues were harvested at indicated time points and stored at -20 ^oC until analysis. Upon thawing, tissues (100mg of tissue/ml of extraction buffer) were homogenized in PBS containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride – PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KI aprotinin A), 0.05% Tween 20 and 0.5% bovine serum albumin (BSA). Samples were homogenised in bead mill homogeniser (TissueLyser LT; Qiagen). Samples were then centrifuged for 10 min at 10,000 rpm, and the supernatant was stored at -20 ^oC until use in enzyme-linked immunoassays (ELISA). Samples were diluted at 1:2 or 1:3 in BSA/PBS, and cytokine levels were assessed by ELISA according to the manufacturer instructions.

I hope this helps you in your research.

All the best,

Dr Michelle Sugimoto

REFERENCES

Amaral DC, Rachid MA, Vilela MC, Campos RD, Ferreira GP, Rodrigues DH, et al. (2011). Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice. J Neuroinflammation 8: 23.

Coelho FM, Pinho V, Amaral FA, Sachs D, Costa VV, Rodrigues DH, et al. (2008). The chemokine receptors CXCR1/CXCR2 modulate antigen-induced arthritis by regulating adhesion of neutrophils to the synovial microvasculature. Arthritis Rheum 58: 2329-2337.

Vieira AT, Galvao I, Macia LM, Sernaglia EM, Vinolo MA, Garcia CC, et al. (2017). Dietary fiber and the short-chain fatty acid acetate promote resolution of neutrophilic inflammation in a model of gout in mice. J Leukoc Biol 101: 275-284.