Why add phenol red in trypsin? - (Apr/27/2005 )
QUOTE (perneseblue @ Jul 29 2007, 02:41 AM)
No. 
Trypsin is an enzyme that nibbles away at the cell's protein anchorage to the plate. EDTA chelates Mg ions, causing the cell to round up. Both in concert cause a cell to detech from the plate.
Trypsin is an enzyme that nibbles away at the cell's protein anchorage to the plate. EDTA chelates Mg ions, causing the cell to round up. Both in concert cause a cell to detech from the plate.
Dear All,
Your are all right that Phenol in Trypsin/EDTA is an indicator of pH. However there is a simpler reason for using it in Trypsin solution.
Basic passaging requires washing the monolayer with PBS Dulbecco's (W/O Phenol), you then add your trypsin/EDTA (Versene) WITH PHENOL RED to detach the cells. You can buy Trypsin W/O Phenol BUT IT LOOKS EXACTLY THE SAME AS PBS in the flask..............the number of times people have bought Trypsin(W/O Phenol) and added PBS instead, and after 20 minutes complain that their trypsin is noy working.
-Rhombus-
anyway, you should not incubate trypsin too long togather with your cells... that will cause your cells's anchorage protein complete destroyed and your cells will not adhere again. always check you cells, once it rounded up, put in media with FBS to stop the reaction. since you only incubate trypsin for less then 5 min, it shouldn't give you much problem in cells viability.
-sanjiun81-
QUOTE (sanjiun81 @ Jul 31 2007, 01:47 AM)
anyway, you should not incubate trypsin too long togather with your cells... that will cause your cells's anchorage protein complete destroyed and your cells will not adhere again. always check you cells, once it rounded up, put in media with FBS to stop the reaction. since you only incubate trypsin for less then 5 min, it shouldn't give you much problem in cells viability.
Hi there
I have not done static flask work for 8 years now (been doing suspension culture) and am struggling to recall how to do it!! What I can't remember is do you remove the medium then add the trypsin to the flask, incubate for 5 min, and then add your fresh medium straight into the cells + trypsin.EDTA. OR must you remove the medium, then add the trypsin and remove it off the cells after abotu 30s and then incubate the cells with the trypsin residue, then add fresh medium when the cells look like they are detaching (about 5 min later).
I don't want to leave the Trypsin in the mix with the fresh medium if it is going to affect the attachment properties of the cells as that is NB in the work I am doing right now.
Also, with normal CHO cells, what seeding concentration would you use if you are only subculturing every 3-4 days (ie like in reasearch when you can sub on a Mon and a Friday). In suspension culture we'd seed at 2e5cells/ml. I find My cells are growing quite fast and I am havign to subculture more than every 4 or so days and this is wasting expensive medium. Maybe I should reduce the amount of serum I am culturing in? 10% at the moment.
Thanks
DC1
-DC1-
stilll yet, wondering why some manufacturers put in phenol red in medium and trypsin EDTA since they can just neglect the addition...right???why is that??
phenol red can interfere with absorption assays. for instance some tetrazolium salt assays will turn your medium red and their absorption wavelength could be similar to the one of phenol red. in this case using w/o phenol red medium improves the sensitivity of the assays.
-coastal-
QUOTE (DC1 @ Jan 17 2008, 04:12 AM)
Hi there
I have not done static flask work for 8 years now (been doing suspension culture) and am struggling to recall how to do it!! What I can't remember is do you remove the medium then add the trypsin to the flask, incubate for 5 min, and then add your fresh medium straight into the cells + trypsin.EDTA. OR must you remove the medium, then add the trypsin and remove it off the cells after abotu 30s and then incubate the cells with the trypsin residue, then add fresh medium when the cells look like they are detaching (about 5 min later).
I don't want to leave the Trypsin in the mix with the fresh medium if it is going to affect the attachment properties of the cells as that is NB in the work I am doing right now.
I have not done static flask work for 8 years now (been doing suspension culture) and am struggling to recall how to do it!! What I can't remember is do you remove the medium then add the trypsin to the flask, incubate for 5 min, and then add your fresh medium straight into the cells + trypsin.EDTA. OR must you remove the medium, then add the trypsin and remove it off the cells after abotu 30s and then incubate the cells with the trypsin residue, then add fresh medium when the cells look like they are detaching (about 5 min later).
I don't want to leave the Trypsin in the mix with the fresh medium if it is going to affect the attachment properties of the cells as that is NB in the work I am doing right now.
we remove media and wash it once with PBS and add trypsin for 3-5 min. Now add media with serum to stop trypsin and resuspend cells and split them according to your needs. Actually the trysin is inhibited by serum in the media and it is diluted atleast 20 fold when you split and seed the cells.
One removes media and washes so that you get rid of the serum which can inhibit trypsin activity.
-scolix-