High sensitivity mouse cardiac troponin 1 elisa kit

Serum cardiac-specific troponin-I (cTnI) was determined by using the high-sensitivity mouse cardiac troponin-I ELISA kit from Life Diagnostics, Inc (West Chester, PA). Briefly, serum was isolated from mock infected and P.a. infected mice, as described earlier, and used for the assay as directed by manufacturer. All assays were performed with 8 mock infected and 9 P.a. infected animals, and the values obtained were plotted as mean ± SD. Significance was P<0.005.

The mouse MI model was induced by permanent ligation of the left coronary artery, as previously described 22. In brief, mice were anaesthetized with 2% isoflurane, intubated and mechanically ventilated (120 strokes/min, 200 μl stroke volume, Hugo Sachs Elektronik Minivent, Cambridge, UK). After a left‐sided thoracotomy, MI was induced by ligating the proximal portion of the left coronary artery using an 8‐0 PROLENE suture (Ethicon, Somerville, NJ, USA). The chest was then closed in layers and the pneumothorax was evacuated. Buprenorphine (0·05 mg/kg) was given subcutaneously for perioperative analgesia, in addition to 1 ml 0.9% saline for rehydration. Twenty‐four hours after performing coronary artery ligation, a tail blood sample (30 μl) was collected (in 3·2% citrate buffer) for assay of troponin I by enzyme‐linked immunosorbent assay (ELISA) [Life Diagnostics (West Chester, PA, USA) high sensitivity mouse cardiac troponin‐I ELISA kit, according to the manufacturer’s instructions] to assess the size of injury.

MI was induced by coronary artery ligation (CAL), as previously described (8 (link), 23 (link)), in Hsd11b1^−/− mice, in age matched C57Bl/6 mice (wild type [WT]), in Hsd11b1^CVCre+, and in Hsd11b1^CVCre− mice. Briefly, after injection of analgesic (buprenorphine, 0.05 mg/kg, sc) and intubation for mechanical ventilation (120 strokes/min, 200-μL stroke volume; HSE-Harvard MiniVent), the chest was opened at the fourth intercostal space, and the pericardium was removed to allow ligation of the left anterior descending coronary artery (6-0 PROLENE suture; Ethicon). Successful ligation was confirmed by blanching of the left ventricle (LV), and the chest was closed using a 5-0 MERSILK suture (Ethicon), ensuring that no air remained in the chest cavity. The skin was closed using 9-mm stainless steel autoclips (Harvard Apparatus). After surgery, 1.5-mL sterile saline (0.9%, sc) was administered to aid recovery. Twenty-four hours after induction of MI, a blood sample (45 μL) was collected from the tail vein into a tube containing sodium citrate buffer for assay of troponin I by ELISA (Life Diagnostics High Sensitivity Mouse Cardiac Troponin-I ELISA kit) to assess the extent of injury (24 (link)).

All procedures were performed in compliance with the UK Home Office “Guidance on the Operation of the Animals” (Scientific Procedures) Act, 1986 and institutional guidelines. Nox4 KO mice (on a C57BL6 background) were described previously (Zhang et al, 2010). Heart ischemia/reperfusion (I/R) injury was assessed in hearts of Nox4 KO animals and matched WT littermates. Hearts were perfused with a modified KHB buffer on a Langendorff system as previously described (Stenslokken et al, 2009). Hearts were stabilized for 20 min before being subjected to 25 min global ischemia and 30 min of reperfusion. In some hearts, xestospongin C was added to the perfusion buffer at a final concentration of 2 μmol/l during the pre‐ischemic stabilization period. Coronary effluents were collected for troponin I measurements. Hearts were snap‐frozen after 30 min reperfusion for subsequent immunoblotting analyses. Cardiac troponin I was measured using a high sensitivity mouse cardiac troponin I ELISA kit (Life Diagnostics Inc., West Chester, PA, USA).