Establishing Immortalized Brown and White Preadipocyte Cell Lines from Young and Aged Mice.

Studying adipogenesis and adipocyte biology requires the isolation of primary preadipocytes from adipose tissues. However, primary preadipocytes have a limited lifespan, can only undergo a finite number of divisions, and often lose their original biological characteristics before becoming senescent. The repeated isolation of fresh preadipocytes, particularly from young pups or aged animals, is costly and time consuming. Immortalization of these cells offers a solution by overcoming cellular senescence and maintaining proliferative capacity, allowing for long-term studies without the continuous need to isolate new cells from animals. Immortalized cell lines thus provide a consistent and reproducible experimental model, significantly reducing variability across different animals. However, successfully establishing immortalized preadipocyte cell lines presents challenges, including selecting appropriate adipose tissue depots, isolating primary preadipocytes, and choosing an effective immortalization strategy. In this article, we present optimized protocols and share first-hand experiences establishing immortalized brown and white preadipocyte cell lines from young and aging mice. These protocols offer a valuable resource for researchers studying adipogenesis and metabolism. © 2024 Wiley Periodicals LLC. Support Protocol 1: Retrovirus production Basic Protocol 1: Isolation and culture of primary brown and white preadipocytes from mouse interscapular brown adipose tissue (iBAT) and subcutaneous white adipose tissue (sWAT) in the same region Basic Protocol 2: Immortalization of mouse brown and white preadipocytes Basic Protocol 3: Selection of immortalized preadipocytes Basic Protocol 4: Selection of single-cell clones of immortalized mouse preadipocytes Basic Protocol 5: Single-cell sorting in a 96-well plate using a flow cytometer for the selection of single-cell clones of immortalized preadipocytes Support Protocol 2: Cryopreservation of immortalized mouse preadipocytes Support Protocol 3: Thawing and culture of cryopreserved immortalized mouse preadipocytes Support Protocol 4: Subculture and expansion of immortalized mouse preadipocytes Basic Protocol 6: Differentiation of immortalized mouse brown and white preadipocytes Support Protocol 5: Identification of differentiated white and brown adipocytes.