Dispersing Agents For Cell Cultures
March 5, 2008 at 1:16 am #9218FusionVisionaryParticipant
The question I have is about dispersing agents. What is the most "efficient"/"best" dispersing agent?
And what is the most appropriate dispersing agent for cells? (This is all with respect to the P19-Keep in Mind).
So far, what i figure is that Trypsin and EDTA, is the most common dispersing agent. From what i understand it
is used most often, so i might be the most appropriate since everyone, researchers and we students are using it.
Trypsin more or less is a serine protease which is used in digesting (?), which digests certain proteins. So with
respect to growing cells, it will cause the extracellular and membrane proteins to be digested hence dispersing them and breaking them up from chunks. Long term, over exposure is said to kill the cells, but (i think), it just errodes the membrane so much that it hinders growth since they must regrow the surface proteins before going past the lag stage. (?) As for EDTA, it expresses some toxic properties as well, but is also used as a dispersing/anticoagulant.
So far i found very little information on other dispersing agents. Found a bit on Disodium/EDTA, and other stuff like acids. I was wondering if anyone know any specific (more or less commonly/known) dispersing agents which i can use to compare effectivity?
I still havn’t managed to find much. I was reviewing some past experimental notes, and we did a similar lab where we tested dispersion abilities of random agents, such as Collagenase, Accutase, HBSS, EDTA, Trypsin/EDTA, and Trypsin themselves. Im looking any other possible "applicable" agents.
March 5, 2008 at 5:13 pm #82513MrMisteryParticipant
What is wrong with trypsin?
We use trypsin in dispersing the cells from the flask in solution. The procedure works by adding trypsin(the concentration depends on the line you’re working with – some cells have a much more compact matrix and therefore require a higher concentration to disperse) to the flask, then incubating it at 37 celsius(the optimum temperature for trypsin) for 5-7 minutes(the exact time depends on the the line again). After the cells have lost their adhesion to the flask, the trypsin is inactivated by adding some culture medium with fetal serum. Now, if you don’t inactivate the trypsin the cells will not all die, however some of them will. Also, they will adhere much harder and take much longer until division resumes. The method takes under 20 minutes if you have a skilled hand and has very good results, with minimum stress to the cell. I don’t see why you are looking for others. What problems have you had?
March 5, 2008 at 5:40 pm #82517FusionVisionaryParticipant
I think you misunderstood me, I’m supposed to write a paper on dispersing agents and their
properties on the P19 *which we are using*. The reference to the lab was just an example of
some other agents that i had contact with. My question basically is just two things, the first
is (would trypsin be the most efficient dispersing agent? maybe why?) and what are some
other dispersing agents used for the p19 cell line. Also what would be an example of a very
effective dispersing agent (not the most appropriate, since it would be very destructive)?
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