It seems to make a huge difference if you use pre-natal or d1 astrocytes and microglia for primary cultures. They grow faster and are easy to culture but those do not refelct the adult cell behaviour.
The main question you should ask is to what end do you want to prepare these cells for? There are currently three ways to purify astrocytes from CNS, each with their own positives and negatives:
1. MD 'shake-off' method - little hands on time, lots of cells, however they do not retain their in vivo gene profiles and likely are already reactive (Zamanian et al., 2012)
2. magnetic bead separation - quick, lots of cells, however less pure than other separation methods
3. immunopanning - highly pure populations of cells which retain their resting in vivo gene profiles (Foo et al., 2011), however very hands on and time consuming process to generate
The additional problem of myelination in animals as they age makes the separation of CNS cell types quite difficult, with many of these cells able to be purified for acute genotyping experiments, however not having great viability when cultured for any period of time. In our experience, the most important factors to consider when purifying CNS cells are:
1. time to dissect - faster is better, and the more you can remove from your samples the better your separation will be (ie. blood vessels, other regions not of interest)
2. digestion (both enzymatic and mechanical) - too little and they wont come apart, too much and the viability drops rapidly
3. culture media - when growing the cells it is important to consider what effect your growth media will have on your cells. The difference between media supplemented with serum (not normally in the CNS) or defined growth factors such as HBEGF is profound, and could cause artefacts in your final cell populations.
The main question you should ask is to what end do you want to prepare these cells for? There are currently three ways to purify astrocytes from CNS, each with their own positives and negatives:
1. MD 'shake-off' method - little hands on time, lots of cells, however they do not retain their in vivo gene profiles and likely are already reactive (Zamanian et al., 2012)
2. magnetic bead separation - quick, lots of cells, however less pure than other separation methods
3. immunopanning - highly pure populations of cells which retain their resting in vivo gene profiles (Foo et al., 2011), however very hands on and time consuming process to generate
The additional problem of myelination in animals as they age makes the separation of CNS cell types quite difficult, with many of these cells able to be purified for acute genotyping experiments, however not having great viability when cultured for any period of time. In our experience, the most important factors to consider when purifying CNS cells are:
1. time to dissect - faster is better, and the more you can remove from your samples the better your separation will be (ie. blood vessels, other regions not of interest)
2. digestion (both enzymatic and mechanical) - too little and they wont come apart, too much and the viability drops rapidly
3. culture media - when growing the cells it is important to consider what effect your growth media will have on your cells. The difference between media supplemented with serum (not normally in the CNS) or defined growth factors such as HBEGF is profound, and could cause artefacts in your final cell populations.
we already use magnetic beads with prior myelin removal - all fine - good amount of viable cells immediately after preparation. Its about culturing the cells
I have attached a protocol adapted from Sato et al. 2009. It is for the brain, but the spinal cord is processed in the same manner. This is done with adult mice (>10wks old).
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