Using whatever EDTA should be OK, if it is soluble and doesn't change the pH.
thank u so much. But I make the solution with 0.2g EDTA.2Na in 1l PBS 1x. The trypsin activity is stronger than normal and the cell detach within only 2 minutes at room temperature. After centrifugation at 1250rpm/5m, I resuspend in growth medium and culture, but the cells are almost death.
If the trypsin is too active, you can either dilute it more with PBS or add 10% serum containing media to the trypsinized cells before the centrifugation step.
I agree with Steingrimur, u can either dilute it with PBS, or u can use less quantity as 0.5ml instead of 1 or 2ml, and add media with FBS directly to stop the trypsinization.
Today, I try with commercial trypsin solution. I also delute in PBS 1X and use only 200ul trypsin + 800ul PBS for 25cm2 flask. The cells detach within only 1minute. After that I add 1ml media with 10% FBS to stop the trypsinization. Centrifuge at 1250rpm/5m and resuspend in media with 10% FBS. But the result like before, the cells are almost death. Here are the images of the cells before and after trypsinization:
http://www.vienngonnguquocte.edu.vn/data/before.JPG
http://www.vienngonnguquocte.edu.vn/data/after.JPG
Hi Long, I saw the pictures as u said the cells are almost die, to solve this problem u can trasfer the trypsinized cells without centrifugation into new flasks, because I have faced this problem before when I was using NCI H1650 cell line, but after I trasfered it without centrifugation it worked well.
try this and good luck.
Other thing is the problem is from your cells not from trypsin, Becasue some cells are detach or die without known reason, so u have to check this trypsin in another cell line or another batch of your cell, or use another trypsin (Commercial) to be sure the problem is not from your cells.
thank you so much. I have tried with your suggestion but got the same result. I have found out that our incubator has problem despite the parameters display correct.
Trypsin solution for primary cultures was prepared by dissolving 2.5g of trypsin in 100 ml of PBS and stirred continuously on a magnetic stirrer at room temperature. Then sterilized by Nalgen filter 0.22μm, and stored at 4C°.
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