Chest

A schematic overview of the myocyte isolation procedure is shown in Figure 2. An expanded description of the procedure, accompanied with images and videos, and complete materials list is available in the Online Data Supplement, alongside full details of additional methods applied in this study (Appendix A-ix). All animal work was undertaken in accordance with Singapore National Advisory Committee for Laboratory Animal Research guidelines. Relevant national and institutional guidelines and regulations must be consulted before commencement of all animal work.
Buffers and media were prepared as detailed in Appendix D. EDTA, perfusion, and collagenase buffers were apportioned into sterile 10 mL syringes, and sterile 27 G hypodermic needles were attached (Online Figure IA).
C57/BL6J mice aged 8 to 12 weeks were anesthetized, and the chest was opened to expose the heart. Descending aorta was cut, and the heart was immediately flushed by injection of 7 mL EDTA buffer into the right ventricle. Ascending aorta was clamped using Reynolds forceps, and the heart was transferred to a 60-mm dish containing fresh EDTA buffer. Digestion was achieved by sequential injection of 10 mL EDTA buffer, 3 mL perfusion buffer, and 30 to 50 mL collagenase buffer into the left ventricle (LV). Constituent chambers (atria, LV, and right ventricle) were then separated and gently pulled into 1-mm pieces using forceps. Cellular dissociation was completed by gentle trituration, and enzyme activity was inhibited by addition of 5 mL stop buffer.
Cell suspension was passed through a 100-μm filter, and cells underwent 4 sequential rounds of gravity settling, using 3 intermediate calcium reintroduction buffers to gradually restore calcium concentration to physiological levels. The cell pellet in each round was enriched with myocytes and ultimately formed a highly pure myocyte fraction, whereas the supernatant from each round was combined to produce a fraction containing nonmyocyte cardiac populations.
CM yields and percentage of viable rod-shaped cells were quantified using a hemocytometer. Where required, the CMs were resuspended in prewarmed plating media and plated at an applicationdependent density, onto laminin (5 μg/mL) precoated tissue culture plastic or glass coverslips, in a humidified tissue culture incubator (37°C, 5% CO₂). After 1 hour, and every 48 hours thereafter, media was changed to fresh, prewarmed culture media.
The cardiac nonmyocyte fraction was collected by centrifugation (300g, 5 minutes), resuspended in fibroblast growth media, and plated on tissue-culture treated plastic, area ≈ 23 cm² (0.5× 12-well plate) per LV, in a humidified tissue culture incubator. Media was changed after 24 hours and every 48 hours thereafter.

We enrolled participants from August 2002 through April 2004; screening took place from August 2002 through September 2007. Participants were followed for events that occurred through December 31, 2009 (Fig. 1 in the Supplementary Appendix, available at NEJM.org).
Eligible participants were between 55 and 74 years of age at the time of randomization, had a history of cigarette smoking of at least 30 pack-years, and, if former smokers, had quit within the previous 15 years. Persons who had previously received a diagnosis of lung cancer, had undergone chest CT within 18 months before enrollment, had hemoptysis, or had an unexplained weight loss of more than 6.8 kg (15 lb) in the preceding year were excluded. A total of 53,454 persons were enrolled; 26,722 were randomly assigned to screening with low-dose CT and 26,732 to screening with chest radiography. Previously published articles describing the NLST^{10 (link),12 (link)} reported an enrollment of 53,456 participants (26,723 in the low-dose CT group and 26,733 in the radiography group). The number of enrolled persons is now reduced by 2 owing to the discovery of the duplicate randomization of 2 participants.
Participants were enrolled at 1 of the 10 LSS or 23 ACRIN centers. Before randomization, each participant provided written informed consent. After the participants underwent randomization, they completed a questionnaire that covered many topics, including demographic characteristics and smoking behavior. The ACRIN centers collected additional data for planned analyses of cost-effectiveness, quality of life, and smoking cessation. Participants at 15 ACRIN centers were also asked to provide serial blood, sputum, and urine specimens. Lung-cancer and other tissue specimens were obtained at both the ACRIN and LSS centers and were used to construct tissue microarrays. All biospecimens are available to researchers through a peer-review process.