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Updated: Aug 27, 2026
Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes
Published on: December 27, 2014
Localization of serum-derived alpha 2 macroglobulin in cultured cells and decrease after Moloney sarcoma virus
Abstract:
NRK cells and many other cultured fibroblasts were found to contain the protease inhibitor, alpha 2 macroglobulin (alpha 2M). This alpha 2M is present as a result of uptake of alpha 2M from the calf serum in the culture medium. Some of this alpha 2M is released back into the medium. In radiolabeling experiments with 14C-amino acids, no radioactivity was detected in intracellular or extracellular alpha 2M. Fluorescence microscopy of fixed cells using rhodamine-labeled antibodies indicated that alpha 2M is present in vesicular organelles different from primary lysosomes. Fluorescence microscopy of living cells shows that rhodamine-labeled alpha 2M (rhodamine-alpha 2M) is taken up into similar structures. Of the many cell lines examined, Moloney sarcoma virus-transformed cells had the lowest amounts of alpha 2M. Some of the effects of serum on the behavior of cultured cells could be a consequence of inhibition of cellular proteases by alpha 2M.
Insights
Cultured fibroblasts absorb alpha 2 macroglobulin (alpha 2M) from serum, storing it in vesicles distinct from lysosomes. This protease inhibitor may influence cell behavior by inhibiting cellular proteases.
Area of Science:
- Cell biology
- Biochemistry
- Protease inhibition
Background:
- Cultured fibroblasts, including NRK cells, contain alpha 2 macroglobulin (alpha 2M).
- Alpha 2M is acquired from calf serum present in the cell culture medium.
- A portion of the internalized alpha 2M is subsequently released back into the medium.
Purpose of the Study:
- To investigate the intracellular localization and origin of alpha 2 macroglobulin (alpha 2M) in cultured fibroblasts.
- To determine if alpha 2M synthesized by cells is released into the culture medium.
- To explore the potential role of alpha 2M in mediating serum effects on cell behavior.
Main Methods:
- Radiolabeling experiments using 14C-amino acids to trace protein synthesis.
- Fluorescence microscopy with rhodamine-labeled antibodies to visualize alpha 2M in fixed cells.
- Fluorescence microscopy of living cells to observe the uptake of rhodamine-labeled alpha 2M.
Main Results:
- Intracellular and extracellular alpha 2M showed no detectable radioactivity, indicating it is not synthesized by the cells.
- Alpha 2M was localized in vesicular organelles distinct from primary lysosomes.
- Rhodamine-labeled alpha 2M was observed to be taken up into similar vesicular structures in living cells.
- Moloney sarcoma virus-transformed cells exhibited the lowest levels of alpha 2M.
Conclusions:
- The alpha 2 macroglobulin found in cultured fibroblasts originates from the culture medium's serum.
- Alpha 2M is sequestered within specific intracellular vesicles, not lysosomes.
- Serum-derived alpha 2M may modulate cellular functions through protease inhibition, potentially affecting cell behavior.
