Trypsin/EDTA

Hello there folks,

I use for some endothelial cells trpsin/edta and according to the
protocol I have to pipette in 5 ml trypsin/edta and then immediately
remove 4.5 ml and leave the rest for a couple of minutes. Does anyone
know why? Isn't this simply a waste of trypsin?

Furthermore, is there any place where I can find detailed info about
trypsin in particular? Thanks for any info.

Cheers,
Lara

In article <[Only registered users see links. ]>, Lara <[Only registered users see links. ]> wrote:

Not immediately. Slosh the dish few times and *then* immediately
remove.


No, it's not. There is always some medium left - almost 0.5 ml
from 10 cm dish, typically. So it makes a huge difference
whether you dilute it with 0.5 ml or 5 ml of trypsin/EDTA.
Remember that most media contain serum and serum contains
powerful trypsin inhibitor - you want to add enough trypsin to
titrate it out. Likewise, medium contains Mg and Ca - you want
to add enough EDTA to chelate them or cells won't detach.
You could just wash with EDTA and add little trypsin but the
cost of doing in sterile pipettes and time exceeds the cost of
the crude trypsin preparation used in cell culture.


Any of the hundreds of the cell culture practical guide books.
Some cell lined don't even require trypsin - just EDTA is
enough.

DK

[Only registered users see links. ] (DK) wrote in
news:BcXck.350$[Only registered users see links. ]:

I agree with DK.

On the other hand, we generally use collagenase, a rather drude
preparation with trypsin as a major contaminant. Because EC are generall
grown on a gelatin coating, you'll need a bit more enzyme activity than
when detaching cell lines from just plastic. It may even depend on how
you lay down the gelatin, coat 1 hr at RT, vs overnight at 4C.


--
Best regards
Han
email address is invalid

Dear DK and Han,

thanks so much for your helpful feedback. Indeed, now I understand it
is not a waste of either Trypsin or EDTA. With responses like this
newsgroups get another dimension in science. Thank a lot again!

Yours,
Lara








On Jul 8, 5:24*pm, Lara <[Only registered users see links. ]> wrote:

Lara wrote:
It most certainly is a waste of trypsin/EDTA. If what DK says is true,
and the extra solution is to dilute serum-containing medium, you can
achieve the same goal by washing the plate once with PBS and then adding
a smaller volume of trypsin. Here is what I do for 10 cm plates or 75
cm2 flasks: aspirate medium, wash once with 10 ml PBS, aspirate, add 1
ml trypsin/EDTA and place in the incubator for 5 min, or until cells
detach. I have never had a problem.

In article <[Only registered users see links. ]>, Kyle Legate <[Only registered users see links. ]> wrote:

Which is certainly true: it is an undeniable fact that serum contains
alpha2-macrglobulin and medium contains ~ 2 mM of Mg2+ and
Ca2+.


Yes, you repeated exactly what I said. That extra step is perfectly
OK and serves exactly the same purpose as adding lots of trypsin
and then aspirating most of it. As I wrote earlier:
"You could just wash with EDTA and add little trypsin but the
cost of doing it in sterile pipettes and time exceeds the cost of
the crude trypsin preparation used in cell culture."

DK

"DK" <[Only registered users see links. ]> wrote in message
news:4Ahdk.2981$[Only registered users see links. ]...

I normally just wash with a bit of PBS and then put a small amount of
trypsin, like Kyle said, but that's because I usually deal with only a few
flasks at any one time. When you are facing several stacks containing a
hundred or so dishes, adding extra trypsin to dilute remaining medium and
removing most of it is a much more palatable approach...

Jose

DK wrote:

I'm not denying it.

DK wrote:

I'm not denying it. Clearly adding extra trypsin and then removing it
dilutes inhibiting factors, but this practice was probably first
suggested by a company to sell more product. It is not the better way.
Washing with PBS is preferable because it more effectively removes the
inhibitors (as you don't leave any behind in the flask when you aspirate
all the PBS), and washing with PBS first removes dead cells and debris
much more effectively.

> Washing with PBS is preferable because it more effectively removes the

I am not denying any of what has been written so far but you have to
apologize if I might ask you what constitutes chemically the fact that
it is more "effective". The expression more effective seems to me a
bit too broad or if you want too vague. After trypsinization you
neutralize with HBBS and remove so therefore you leave no traces
behind. The question here is not what is more economical but what does
better to the cells and since we all are interested in what is best
for our cells we stand on the same site, right?

Lara