Hi all, I have a set of cell culture samples embedded in 1% agarose. They have been cultured for 14 days. They have been fixed with 4% PFA. I am wondering if I can separate the whole cells from the agarose gel from these fixed samples and test proteomics from both the gels AND cells separately?
Kais Khudhair al Hadrawi
Mechanical Dissociation:Use a spatula or scalpel to carefully scrape the cells off the gel surface. Use a cell scraper or cell lifter to detach cells from the gel surface gently. Enzymatic Digestion: Treat the gel with an appropriate enzyme that can digest the gel matrix without affecting the viability of the cells. For example:Collagenase for collagen-based gels. Trypsin or dispase for protein-based gels. Agarase for agarose gels. Incubate the gel with the enzyme solution at an appropriate temperature and time, following the manufacturer's instructions. After digestion, gently pipette or wash the cells off the gel surface. Dissolution: If the gel is made of a material that can be dissolved, such as agarose or polyacrylamide, you can dissolve the gel to release the cells. For agarose gels, immerse the gel in a buffer solution at an appropriate temperature to dissolve the gel. For polyacrylamide gels, you may need to use a denaturing agent such as SDS or urea to help dissolve the gel. Electrophoretic Transfer: If the cells are embedded within a gel used for electrophoresis, such as a polyacrylamide gel used for protein separation, you can perform electrophoretic transfer to transfer the separated proteins onto a membrane. After transfer, the membrane can be probed with antibodies or stains to detect specific proteins or visualize total protein content. Filtration: For certain types of gels, such as collagen gels used for 3D cell culture, you can use a cell strainer or mesh filter to physically separate the cells from the gel matrix. Cells can be washed through the filter using a buffer solution, while the gel matrix is retained on the filter. Centrifugation:If the cells and gel are suspended in a solution, centrifugation can be used to separate the cells from the gel. Adjust the centrifugation conditions (speed, time, temperature) based on the properties of the gel and cells.
Separating cells from gels can be done using various methods depending on the type of gel and the specific application. Here are some common techniques:
Choose the most appropriate method based on the type of gel, the nature of the cells, and the downstream applications. Always handle cells and gels gently to maintain cell viability and integrity.
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