The identity of the apoptotic cell explains the functional diversity of the efferocytic macrophages
The identity of the apoptotic cell explains the functional diversity of the efferocytic macrophages
Mouse livers were perfused manually, via injection of 20 mL PBS in the vena cava. After removing the gallbladder, the liver was minced and the tissue homogenate incubated with digestion buffer (DMEM, 1 mg/mL Collagenase IV StemCell, 150 U/mL DNaseI, 0.2 M MgCl2, 0.5 M CaCl2) for 45 min at 37°C while shaking. To obtain a single- cell suspension, the cell homogenate was smashed through a 70 µm filter and washed three times with PBS (centrifugation at 300 x g, 5 min, 4°C). The non-parenchymal cells were obtained by collecting the cell supernatant after centrifugation at 50 x g for 4 min at 4 °C. Afterwards, 6 mL 37 % Percoll per liver was added to the non-parenchymal cells and centrifuged for 10 min at 400 x g (dec.1 acc.9), followed by a RBC lysis. Cells were then washed and used either for phenotypical characterization at the LSRII (BD Biosciences) or for cell sorting at the BDAria (BD Biosciences). FACS-sorted cells were washed once with PBS + 0.04% bovine serum albumin and resuspended in PBS. 10,000 cells were used for GEM generation using the 10x V3.1 Single Cell Kit and Chromium Controller (10x Genomics, USA) according to the manufacturer's instructions. Droplet preparation and cell barcoding was followed by reverse transcription. Emulsions were resolved and the cDNA was purified and amplified by PCR. Amplified cDNA was fragmented, end-repaired and size selected using SPRIselect beads. Index primer were used for gene expression library construction.