Polarstar omega microplate spectrophotometer

To assess the impact of loxP site insertions on the functional capacity of expressed IL-1R1s, we evaluated the ability of recombinant IL-1α to increase serum IL-6 expression in vivo, as described by Glaccum et al. [27 (link)]. Briefly, male Il1r1^loxP/loxP mice and their wild type littermates were injected i.p. with either vehicle (saline, 0.1 mL total volume) or 1 μg recombinant IL-1α (0.1 mL/animal of a 10 μg/1 mL solution) (GenScript Piscataway, NJ). Two hrs post-injection, mice were sacrificed by rapid decapitation, trunk blood was collected and allowed to sit for 30 min at room temp. To determine whether excision of Il1r1 alleles by CMV Cre expression results in an elimination of IL-1R1 function, a similar breeding strategy as that described above was utilized to obtain Il1r1^loxP/loxP:CMV Cre and Il1r1^loxP/loxP mice. These mice, in conjunction with Il1r1^-/- animals as negative controls, were treated with IL-1α (1μg) as those described above and two hrs post-injection samples were collected. Samples were then centrifuged at 1500 x g for 10 min prior to being stored at -80°C, IL-6 levels were determined on thawed serum samples by ELISA (Mouse Ready-SET-Go IL-6 ELISA, eBioscience, San Diego, California) according to the manufacturer’s instructions with product quantified using a POLARStar Omega microplate spectrophotometer (BMG Labtech, Ortenberg, Germany).

DNA extraction was performed using the QIAamp DNA mini kit (Qiagen) for both bacterial cultures and total extrapallial fluids. 450 µL were centrifuged at 10,000 g at 4 °C for 10 min. Pellets were deproteinized in a hot dry bath at 56 °C by addition of 180 µL of ATL Buffer supplemented by 20 µL of proteinase K during one hour. DNA extractions were then performed according to supplier instructions. Finally, DNA were eluted in 200 µL of ultra-pure water and stored at −20 °C until use. DNA yield and purity were determined by spectrophotometry (Quantifluor dsDNA kit; Promega, Madison, WI, USA; and POLARstar Omega microplate spectrophotometer; BMG Labtech, Orgenberg, Germany).