papain usage in primary hippocampal cell preperation

[Serra Akinturk]

Is there anyone who uses papain for primary neuron culturing? 

It will be very nice if you inform me about the usage of the enzyme. I guess, I don't use the enzyme in correct way.

Thanks in advance and looking forward to hear from you,

Serra

[Michael Schell]

We use the PAP2 vials from the Worthington Papain Dissociation kit.  The vials can be ordered separately from the kit.  Add 5 ml HBSS to the vial, incubate 10 min at 37 to dissolve, then incubate with the brain pieces (such as intact, dissected hippocampus) for 1 hr at 37, followed by gentle trituration in the presence of DNAse to disperse the tissue.

Michael

Michael J. Schell, Ph.D., CIV, USUHS
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  [phone]
[hidden email]
>>> Serra Akinturk <[hidden email]> 06/01/10 11:11 AM >>>
Is there anyone who uses papain for primary neuron culturing?
It will be very nice if you inform me about the usage of the enzyme. I guess, I don't use the enzyme in correct way.
Thanks in advance and looking forward to hear from you,
Serra


     
Classification:  UNCLASSIFIED
Caveats: None

[Manja Schubert]

Hi Serra,

About the usage of the enzyme: Papain also known as papaya proteinase I
is a  cysteine protease. Its utility is in breaking down  the
extracellular matrix molecules holding the cells together.

I am using papain (102 units) from Worthington Biochemical Corporation.
Cat Number LKoo3178; PDS Kit, Papain Vial 58P10884 (!!! No commercial
interest. Only very satisfied customer!!! :-P)

I using one vial and add 5 ml of  Hanks (HBSS + HEPES; 37C; pH 7.4,
orange) to it shortly before I start to dissect the hippocampi.
Papain needs about 15-20 min to be activated in the incubator.
Dissected hippocampi are placed immediately in ice cold Hanks, after all
are dissected I transfer them in the papain solution and keep them in
the incubator for 15 min, gently shake every 5 min. You can see that the
hippocampi sticking together in a kind of slime network if the papain is
working right.
To stop the digestion you remove really careful the papain solution with
a fire polished pipette and replace it with 5 ml MEM (37C, pH 7.8; Gibco
#11090) containing 10-15% Fetal bovine serum (FBS) and 20 mM Glucose,
mix thoroughly but gently for 2 min....now the papain should be
deactivated by the FBS...important : if there untreated papain's
activity that will lead to complete lysis of the cells.
Last step would be removing MEM and add 5ml of your growth media ( I am
using NBM). Now you start to dissociate the neurons from each other by
pipetting the hippocampi slime ball up and down with a fire polished
pipette until the solution is cloudy. That its...your hippocampal neuron
stock solution is done.

Good luck!

Manja

Serra Akinturk wrote:
> Is there anyone who uses papain for primary neuron culturing?
> It will be very nice if you inform me about the usage of the enzyme. I
> guess, I don't use the enzyme in correct way.
> Thanks in advance and looking forward to hear from you,
> Serra
>

--
Dr. Manja Schubert
University of Bergen
Department of Biomedicine
Nevro Group
Jonas Lies  vei 91
5009 Bergen
Norway
Tel:[phone]
Fax: + 47-55 58 63 60