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): Cut out a 400 µm section of cortex 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) After incubation, cut out a small piece of tissue (max 1 mm3) from the section (I take the outer part of cortex, ca. 600 µm3). 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: 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)) 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? 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). 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? 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?