GSM8194252: CD45.1/.2 2 days after I/R injury, RZ, S106; Mus musculus; RNA-Seq
Heart tissue was dissected into remote and ischemic tissue as described above and minced into small pieces using forceps and a scalpel. When comparing baseline and I/R injury in FACS analysis, basal heart tissue was used for comparison with remote tissue and apical heart tissue for comparison with ischemia tissue. After enzymatic digestion (Collagenase XI 1200 U/mg, Collagenase I 125 U/mg, Hyaluronidase 500 U/mg, DNase I 1836 U/mg; Sigma Aldrich®) for 30 min at 37°C cells were washed and incubated with purified anti-CD16/32 (FcγRIII/II; dilution 1/50) for 10 min. For each sample, ~1000 macrophages were sorted into 75µL of RLT buffer (Qiagen, containing 1% beta-mercaptoethanol), vortexed for 1 min and immediately frozen (-80°C). RNA extraction (RNeasy® Plus Micro Kit, Qiagen), cDNA generation (SMART®-Seq v4 Ultra Low Input RNA Kit, Takara Bio)