Hello all,
When doing cell subculture, many guides suggest to passage cell at ~80% of cell confluence. But if I want to collect treated cell sample, should I collect the cell at ~80% of cell confluence, too? If I wait till 90-100% confluence should get more protein?
I am treating substances in sh-sy5y cell line and want to collect protein sample for western blot analysis. The cell is treated for 24hr in dilution with 1%FBS media.
Cheerio,
I would say 80-90% confluent.
100% confluent or over confluent changes expression of certain genes in the cells through contact inhibition.
Sh-sy5y cells (I have worked with them), grow fast enough and ad that confluent state you have enough protein for western blot.
I harvested the cells by adding boiling (or near boiling since it cools down rapidly) of 10% SDS. This protects your proteins directly and make your sample easy loadable on the gell (just add loading dye and your off). quantification is done by the number of cells you used and by measuring a standard gene that doesn't change expression in western blot for your conditions.
Dear Asa,
I would not go any further than 80-85%, as you might risk that reaching full confluency might trigger intracellular mechanisms that affect protein synthesis and degradation. Cell lines that grow in mono-layers will stop dividing once they covered the available surface, so 80% confluency should still give you plenty of protein lysate to work with.
Moreover, you should plan your experiment so that cells are not too confluent when you harvest them for downstream experiments. If the treatment lasts only 24 hours and does not affect cell proliferation, you can easily split your cells, treat them after 24 hours and collect at 48.
Good luck,
Matteo
contact inhibition blocks expression of many genes involved in transcription regulaiton. Further more you could think of a bigger well (i know it takes more inhibitors/ ligands). For me in most cases 70% confluency workes best
If you need more protein, you should use larger plates or use more of them. As the others have noted, changing confluency will change gene expression.
I would use 80-85 % confluent cells as fully confluent cells behave differently. As Eric Arnoys suggested If you need more protein extract better to use more or larger plate.
It all depends upon the question you are asking. The fact that your cells are in 1% FBS suggests you may be serum starving them which will impact cell cycling, as will density arrest. How you lyse the cells may also have important impact on the results. Some proteins are negatively impacted by boiling in sample buffer and boiling is rarely necessary. If looking for post-translational modifications, e.g., phosphorylation, then freezing cells immediately while lysing may be preferable. If just looking at the amount of a protein that you know is stable to the conditions you are using, I think I would still collect cells at low, medium, and high cell densities at least once just to see the effect. That way you will know for all future experiments.
It depends entirely on the biological process you are studying, and how that process is affected by cell confluence. I suggest that you try WB experiments at varying levels of confluence, and find the optimal conditions for the response you're looking for. Non-optimal confluence conditions could miss or under-estimate the response.
- Badges
- Science method