Lamin A/C expression in hematopoietic cells declines during human aging and constrains atherosclerosis in mice
Lamin A/C expression in hematopoietic cells declines during human aging and constrains atherosclerosis in mice
Aortas from Ldlr-/- mice reconstituted with Lmna-/-, Lmna+/+, Lmna-OE, or Lmnatg BM were thoroughly perfused with PBS to remove blood. The aortic arch and thoracic aorta were dissected, cleaned of perivascular fat and opened longitudinally. Aortas from 3-5 transplant recipients of the same genotype were pooled, with both male and female donors included in each pool to minimize potential sex-related bias. Aortas were digested at 37°C for 15 minutes in DMEM supplemented with collagenase A (6.25 mg/ml, Roche), dispase II (6.25 mg/ml, Roche), DNase I (62.5 μg/ml; Roche), and elastase (1.717 U/ml, Sigma). Cells were treated with 0.25% trypsin-EDTA (Gibco) for 5 minutes at 37°C, pelleted and resuspended in sorting buffer. Purified cells were sorted to filter cells according to size and complexity (FSC, SSC), and then select viable nucleated cells (Hoechst 33342+, TO-PRO-3−) for sorting, discarding dead cells and debris. Cells were recounted and their viability checked on a Countess III cell counter (Thermofisher). Cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics) for single cell isolation and cDNA reverse transcription and amplification Sequencing libraries were created using the Next GEM Single cell 3'Library preparation kit v3.1 (10x Genomics) and indexed using the Chromium i7 Multiplex kit (10x Genomics) following the manufacturer instructions