Human myd88 elisa kit

Cardiomyocytes were treated as described in Section 2.2; after treatment, the cells were harvested and lysed in complete lyses buffer (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 100 mM NaCl, 20 mM NaF, 3 mM Na3 VO4, 1 mM PMSF, and protease inhibitor cocktail). After centrifugation, supernatants were collected and treated to the quantification of MyD88 [Human MyD88 ELISA Kit (ab171341); Abcam, Milan, Italy] and NLRP3 [Human NLRP3 ELISA Kit (OKEH03368); Aviva Systems Biology, San Diego, CA, USA]. For the human MyD88 ELISA, the sensitivity was <10 pg/ml and the range of detection was 156–10,000 pg/ml; for the human NLRP3 ELISA assay, the sensitivity was <0.078 ng/ml and the range of detection was 0.156–10 ng/ml (29 (link)).

After treatments, THP-1-derived macrophages were harvested and lysed by lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 100 mM NaCl, 20 mM NaF, 3 mM Na₃VO₄, 1 mM PMSF and protease inhibitor cocktail). Lysates were then centrifuged at 10,000× g for 5 min, and the supernatants were collected and analyzed for quantification of Myd88 through human MyD88 ELISA Kit (ab171341; Abcam, Cambridge, UK). Briefly, an antibody against MyD88 was pre-coated onto a 96-wellplate and blocked. Standards or samples were added to the wells and incubated for 1 h. After washing, a biotinylated detector antibody specific to MyD88 was added and incubated, followed by washing for 30 min. Avidin-HRP conjugate was then added and incubated and unbound conjugate was washed away. An enzymatic reaction was produced through the addition of TMB substrate, which is catalyzed by HRP, generating a blue color product that changes in yellow after adding acidic stop solution. The optical density of yellow coloration (450 nm) was quantitatively proportional to the amount of MyD88 captured in well. Data were expressed as a percentage of the control.