Suspension Cultured Cell Lysis Protocol
- 7 steps
- 45 mins
- 2 reagents required
Unlike adherent cells, suspension-cultured cells grow in a free-floating environment, presenting unique considerations for effective lysis. This protocol focuses on the critical steps for efficiently collecting and lysing these cells while maintaining the integrity of their intracellular constituents.
Designed to be user-friendly and adaptable, this protocol is ideal for molecular biologists and researchers in related fields. It provides a reliable foundation for various downstream applications, from protein assays to RNA studies, ensuring reproducible and high-quality results.
Reagents Required
| Product | Preparation |
| Phosphate Buffered Saline (PBS) | Use 10X PBS, pH 7.2 (0.2 M Potassium Phosphate, 1.5 M NaCl). Dilute appropriate volume to 1X with molecular biology grade water. |
| RIPA Lysis Buffer | Use 1X RIPA Lysis Buffer or prepare 1X solution with 10X RIPA Lysis Buffer and molecular biology grade water. |
Procedure
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Pellet the cells by centrifugation at 450 x g for 5 minutes.
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Carefully remove the medium from the cell pellet by decanting or aspirating the medium.
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Wash the cells to remove residual medium. Add a volume of sterile PBS equal to the original medium volume. Mix or vortex briefly to resuspend the cells completely. Centrifuge for 5 minutes at 450 x g to pellet the cells and carefully remove wash solution supernatant. Repeat the wash once to remove any other minor contaminants. More washing steps can be done, but two is usually sufficient to remove most of the contaminants.
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After removal of the final wash solution from the cells, add an appropriate volume of RIPA Lysis Buffer (1 mL for 0.5 to 5 x 107 cells). Mix or vortex briefly to resuspend the cells completely. Incubate on ice or in a refrigerator (2°–8°C) for 5 minutes. Vortex briefly to resuspend and lyse residual cells.
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The lysate can either be used immediately or quick-frozen in liquid nitrogen and stored at –70°C for future use. It is best to freeze the lysate before clarification, since the freeze-thaw cycle may cause some proteins to denature and aggregate.
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Clarify the lysate by centrifugation at 8,000 x g for 10 minutes at 4°C to pellet the cell debris.
Note: If a mucoid aggregate of denatured nucleic acids is present, carefully remove it with a micropipette before centrifugation. -
Carefully transfer the supernatant containing the soluble protein to a tube on ice for immunoprecipitation or other analysis.