Cell culture - (Mar/30/2010 )

why do we wash cells in serum free medium before removing the cells from the flask by additon of trypsin?

is it to ensure that all the trypsin inhibitor are washed away from the cell sample so that the trypsin can cause its effect? or is it something else?

hope sumone is able to help me out and thx in advance

Hello, I am new here and I need help with serum starving some cells. I have been trying to serum starve some BRE cells and for some reason they are lifting off the plate( I do not believe they are dead). I wash them with PBS before putting in the serum free media. I have 500 ml of serum free media and it has has 50ml of 1% BSA. When I place the serum free media into the dishes, the cells are fine for a while, but then lift up.

because serum contains a lot of proteins which will interfere with the trypsin (not necessarily inhibit but get in the way).

mdfenko on Mar 30 2010, 07:31 AM said:

however, its been washed by serum free medium so tht shudn't have any serum right?

KB13 on Mar 30 2010, 03:57 PM said:

Correct. You need to wash cells to remove the proteins in serum in order for trypsin to work efficiently. Serum free media is an effective but expensive way to go about this. I just use sterile PBS.

tae on Mar 30 2010, 09:30 AM said:

Usually serum starvation is done with at least 0.5% BSA but I have found some references (including one that works with this same cell line) that uses 0.1% BSA but only for short times 15-24 hours max. Then you get apoptosis. Why do you think your cells aren't dying? Have you done tunnel staining? How long are you trying to serum starve and when do you start to see cells lift off the plate? The only other thing I can think of is that you are stressing your cells if you are going directly from 10% serum to 0.1%. You may need to step them down with over a few days to let them slow down and adjust to lower BSA conditions. Go from 10% to 5% for two days, then 2% for two days followed by your serum starvation.

KB13 on Mar 30 2010, 03:50 PM said:

Answer:

Trypsin is inhibited by Divalent Cations such as Magnesium and Calcium. It is also inhibited by proteins that are contained within FCS/FBS. Thus we use PBS W/0 Ca2+/Mg2+ to wash the cell monolayer. This removes both the protein inhibitors and divalent cations that are in the culture media. What we want is the Trypsin to act as efficiently as possible....this is again why we warm it to 37oC...optimal temperature for most enzymes.
If the Trypsin is on the cells for too long, it can be internalised, causing major damage to vital intracellular proteins. This therefore can reduce viability of the cells dramatically.


Hope this is useful

Kindest regards

Uncle Rhombus

rhombus on Apr 8 2010, 12:50 AM said:

It might pay to clarify that Ca does not inhibit trypsin directly but can make the substrate proteins less susceptible to proteases.