If you talk about cellular enzymatic isolation, you can separate neurons from small cells, rests of dendrites/axon and remaining connective tissues using rate zonal centrifugation in a density gradient. the density gradient, the g force applied and for how long determine the separation you will get.

Yes, the plan is to disrupt the fresh tissue with homogenation and enzymatic digestion. Afterwards it would probably be the best thing to put it on Percoll gradient for separation acording to size. What I would like to find is a protocol suitable for disruption of tissue and for centrifugation with Percoll gradient, so I have a strating point protocol that I have to optimize. The cells I will get will be used for DNA/mRNA isolation. The cell bodies have to be as intact as possible, so we cen determine their morphology.

Thanks.

I got a protocol paper accepted a while ago explaining how to do this on neuronal ganglia; the journal isn't that quick however and it's not online yet. Here is a brief summary of the protocol. Keep in mind that the enzymes and times are designed for ganglia which have connective tissue stronger than brain tissue.

1) dissection and transportation of tissue is done in ice cold HBSS calcium, magnesium and phenol red free.

2) for ganglia I use a short (10 min max, 37 dec C, pH 7.4) first isolation step with collagenase (type 4, 600u/ml, best one is from worthington) and 1mM free calcium (no magnesium or ATP) to break down the strong connective tissue. Skip this for brain tissue and resort to this only if you have undigested tissue at the end.

3) 30 min in presence of activated papain (I use sigma, P4762, 37 deg C, pH 4, 20u/ml). The solution to use for this step is HBSS + 0.4mg/ml L-Cysteine + 1ul/ml EDTA (0.5M stock) + 1.5ul/ml CaCl2 (1M stoock).

4) 40 min in presence of collagenase type 4 (600u/ml) and dispase II (Roche, ref 04 942 078 001, 2.5u/ml) all at 37 deg C, pH 4. The solution you use for this step is Calcium-free, magnesium-free, phenol red-free HBSS. You can increase this step's duration if you get incomplete homogenized solution at the end.

Between each isolation step you spin the tissue at 400g for 2 min in order to remove the supernatant.

At the end of the step 4) add HBSS to the pellet and break down the tissue. I use glass pasteur pipettes with decreasing bores (1mm, 0.5mm, 0.3mm) coated with F12 medium (sigma aldrich ref P4333) to break the tissue: alternative suctions releases.

the HBSS should go milky with no bits of tissue remaining.

the gradient to use would be 20% percoll (sigma aldrich p1644) in L15 medium (sigma aldrich ref L5520). the ratio to use is 1ml of HBSS and cell/debris suspension with 5ml percoll/L15.

carefully pipette your HBSS on top of the percoll/L15 mix and spin it. To start with use 9min at 400g; all the debris and small cells will be trapped at the interface and the big cells will go to the bottom.

You may have to/want to modify the gradient centrifugation step, be carefull to adjust the pH if you use more than 20% percoll; the neurons are quite fragiles, they tolerate 20% percoll in L15 for a short time but higher amount of percoll may be toxic (especially the pH which become quite alkaline)

make sure to wash off the percoll with L15.

Also I use poly-lysine + laminin coating to culture the neurons to get good adherence.

Last point, you will certainly have to modify the protocole depending on the type of neurons you use. Also, neo-nates and young neurons will need some NGF growth factor in their culture medium. you don't have to add NGF for mature neurons.

I hope this skirmish off a protocole will help you

:)

any question don't hesitate

Dear Eric,

Thank you so much for the protocol. I will try it out. However, I have a question regarding the volumes I should use for separate steps. Could you advise me on this as well?

Thanks,

Alja

Dear Alja,

volumes are for 2 to 6 ganglia (it works for all sort of neuronal ganglia, DRGs, vagal, jugular, nodose etc...)

Step 2: 1ml of solution final volume

Step 3: 5 ml of solution final volume

Step4: 5 ml of solution final volume.

Also, agitate the solutions (gently, 4-5 time turning the tube upside down is enough) at regular intervals during the isolation (every 10 min)

Hello Eric,

I ran across this post in my quest for a DRG dissociation protocol that would be applicable to monkey tissues. I've been trying for months to modify protocols designed for rodents. Mostly I've tried different combinations and concentrations of collagenase and proteases. In general the digestion has been incomplete and there is always a lot of myelin, debris, and non-neuronal cells that get in the way of subsequent patch-clamping attempts. Your protocol is very intriguing and I think I will give it a try! Just a couple of questions (because I have a feeling that success is in the details): Do you mince the ganglia into small pieces to start? What is the 1mM calcium solution used in step 2 - do you just add 1mM CaCl to HBSS? And for the Percoll gradient, where exactly do the neurons end up - is it in the pellet, or throughout the lower layer?

Your advice would be much appreciated! Monkeys don't come along very often, and I need to get this working more consistently.

Best!

~lisa