Need help with replicating a dissociation procedure of brain tissue into single cells!

[anonymousnorwegian]

Dear ScienceForums.net,

I have a problem with a method I'm trying to replicate for my PhD-thesis in genetics. My goal is to cut out a small piece of brain tissue (ca. 1Mm³⁾ and dissociate the tissue into single cells in suspension. The method for this is given in the article «A manual method for the purification of fluorescently labeled neurons from the mammalian brain (Hempel et al 2007, availiable at http://pdfcast.org/pdf/hempel-et-al-2007-neuron-dissociation )».

Briefly, the method involves (I use newborn mice):

 

1. Cut out a 400 µm section of cortex
2. Incubate section in an enzyme solution that digests proteins (for example «Pronase»; incubation time is longer for older animals; I incubate at 37 degrees Celcius)
3. After incubation, cut out a small piece of tissue (max 1 mm³⁾ from the section (I take the outer part of cortex, ca. 600 µm^3).
4. Move the small piece to a small 1.5 mL tube. Suck the piece in and out10 times with glass pipettes (going from pipetts with 600, 300 and 150 µm mouths). This should dissolve the piece into single cells.

My problem is that I'm not getting a high amount of cells that are alive. By counting live/dead cell ratios (trypan blue labling for dead cell identification) I'm only getting around 10,000 live cells and 20,000 dead cells pr tissue piece that I cut out (which is around 600 µm³⁾ . I need to get a higher number of live cells for my later application.

In regards to my problem, I have several questions that I'd love any insight/comment on:

1. How many live cells are present in a ca. 600 µm³ piece of cortical tissue (0.6 mg)? I'm only getting ca. 10,000 live cells. (By googling I get that an adult human brain weighs ca. 1300 g and contains ca. 80 billion neurons. This gives ca. 62 000 neurons pr 1 milligram of tissue (ca. 1mm³⁾⁾
2. I am not using the ion-channel inhibitors in my solutions (to reduce neural action potentials + glutamate toxicity) currently. Might this be a major reason for the poor live cell count?
3. The article states that pipetting the small piece should leave the solution cloudy. This does not happen for me. Is it because my piece of tissue (ca. 600 µm³⁾ is too small to cloud my solution (1 mL)?
4. The small piece does not seem to dissolve/disintegrate easily regarless of incubation time (I've tried 30-60 minutes on the newborn mice).
5. The article does not state what temperature to use when incubating with pronase (the proteolytic enzyme). Might it be a problem that I'm extracting the cortex in ice-cold solutions, incubating with pronase at 37 degrees, then using ice-cold solutions again in a 15 min step to remove the pronase, and also in the pipetting step? How does temperatur fluctuations affect cell survival and easy dissociation?
6. Should I do more experiments with regards to optimal enzyme incuabtion time, and optimal pipetting forcefulness/gentleness? How narrow might the time for perfect/successful incubation time be?

[CPG]

I've never done that particular procedure so my advice would be highly speculative. However, I have read a few papers recently from Sascha du Lac's lab where something very similar was done. Sometimes reading the methods of multiple authors who have their own variation on a procedure helps to figure out what's most important for your purposes. It has for me on a few occasions. A couple suggestions:

 

http://www.ncbi.nlm.nih.gov/pubmed/17392422

 

I'm not sure if they've adapted their procedure at all but if they have here's a more recent publication from that lab

 

http://www.ncbi.nlm.nih.gov/pubmed/22674258

 

 

 

Good luck!

[hypervalent_iodine]

I'll preface this by saying that in no way am I to be considered a biologist,so my questions may seem a little dim.

 

My problem is that I'm not getting a high amount of cells that are alive. By counting live/dead cell ratios (trypan blue labling for dead cell identification) I'm only getting around 10,000 live cells and 20,000 dead cells pr tissue piece that I cut out (which is around 600 µm³⁾ . I need to get a higher number of live cells for my later application.

 

 

In regards to my problem, I have several questions that I'd love any insight/comment on:

• How many live cells are present in a ca. 600 µm³ piece of cortical tissue (0.6 mg)? I'm only getting ca. 10,000 live cells. (By googling I get that an adult human brain weighs ca. 1300 g and contains ca. 80 billion neurons. This gives ca. 62 000 neurons pr 1 milligram of tissue (ca. 1mm³⁾⁾

 

Human brains and mouse brains are not the same in terms of neuronal packing density and furthermore, I would think that packing density varies with the section of the brain. A quick search of mouse cortexes gives this: http://www.ncbi.nlm.nih.gov/pubmed/2778101

 

So, still very high compared to your results. Could the closer packing density of mouse neurons be affecting your cell count estimate? I do not know how you go about doing such things, so I don't know how accurate they are, etc.

 

At a guess, and this is very much an uninformed guess, it might be possible that your cells are sticking to your pipette and you are losing them in that way. Are you able to use low binding plastic pipette tips at all? I don't know if they'd be any better, but someone here might be able to tell you.

 

 

 

• The article states that pipetting the small piece should leave the solution cloudy. This does not happen for me. Is it because my piece of tissue (ca. 600 µm³⁾ is too small to cloud my solution (1 mL)?
• The small piece does not seem to dissolve/disintegrate easily regarless of incubation time (I've tried 30-60 minutes on the newborn mice).

 

These two thing could be very related. Cloudy would mean that you have something going into suspension, so if your sample won't break up, that may be why you don't see cloudiness.

 

I couldn't comment on the rest as I have no experience in the area. These were just some thoughts I had upon reading your post and may not be meaningful at all.