Hi,
Can any fellow please share his/her experience that how to get live neuronal cell population. I am injecting Alexa conjugated with my neuronal cell specific peptide and i want to see the uptake by neuronal cells in different regions of adult rat brain (cortex, hippocampus etc) by FACS. Last time (attach file) i tried by digesting the isolated regions with collagenase 1 (2mg/mL) and then washing and fixing the cell pellet. But i could not distinguished between neuronal and glia cell population as i do not have cell specific antibody. Please share any simple protocol.
Irfan,
Do you have access to any fluorescent microscopy instrumentation rather than flow? If you are not able to obtain additional markers that can differentiate between neurons and glia (GFAP), then you might possibly be able to rely on the anatomical arrangement which would remain intact in a brain slice, as opposed to flow where you have to dissociate everything. This still might not completely solve your problem as having something like a fluorescent marker for nuclei (propidium iodide, DAPI) is also very handy for almost all preparations like this.
Also you mention that you'd like "live neuronal cells", but then describe a method where you fixed your isolated cells prior to flow. Can you clarify whether getting live cells is important?
JB
Dear Jeffrey,
For a moment i need only total population of brain cells. I am worry about my single cell suspension protocol only. This time i am injecting Cy5 instead of Alexa. Yah i have fluorescence microscope and can do brain tissue sectioning. Can i see Cy5 transfected brain cell by flourescence machine as i do not have confocal machine.
Dear Irfan, you have a good point to Jeffrey's clever suggestion. With thick brain slices (which best preserve the brain morphology: pre-fixation and vibratome cutting) a confocal microscope is absolutely necessary. Alternatively you can try to freeze the dissected brain on powdered dry ice in Tissue-Tek and cut at a cryostat at 10 um thickness, which is approximately the average size of a neuronal cell, with short post-fixing of the slices. Morphology is not very well preserved and cytoplasmic membranes are broken, peptides might flow out, but imaging is easier, so it is a possibility. To detect Cy5, in any case, you need an infrared filter (excitation around 640 nm, emission around 670 nm: check the Chroma spectra viewer).
In your situation, in my opinion it would be best to label neurons with a specific marker (MAP2, for example) for your FACS analysis. You are right, it is almost impossible to get rid of astrocytes, they are in average 50-60% of brain cells. And forward/sideward scatter does not help much, since neurons in the brain are very different in morphology.
Yes, I'll echo what Pietro said and say that confocal would be the ideal platform for the work you are suggesting. The alternative of using very thin frozen sections comes along with the potential disadvantages called out above, plus your need to resolve a fluorescent signal. So the battle come down to slicing them thin enough, but still retain enough dye to emit a signal that you can detect. Going back to your Alexa dye, which may be brighter than the Cy5 might help.
And if it comes out that you are indeed stuck on flow, you may have to come up with a minimal panel of markers to differentiate cells in order to get useful information.
Thanks guys for kind suggestion. Still i could not valuable remarks about making single cell suspensions especially digestion (which enzyme is best) ...
We have the best results in our laboratory by using a dounce homogenizer to make single cell suspensions without having to use any digesting enzyme. I just process brain tissue in DMEM media (with glucose and L-glutamine). We use this method for CNS tissue such as brain or brain stem samples, then wash the homogenate through a 40 or 70 micron filter.
For your cell sorting, you could exclude CD45 positive cells. This will not only exclude your microglia (CD45lo-mid expressing) but also any potentially contaminating leukocytes or myeloid cells (CD45 hi expressing cells). (We find that in naive animals, there are usually a small pool of immune cells even after perfusion with PBS.) You could then exclude your astrocytes with GFAP. I'm not sure about oligodendrocytes, but you could see if there is another marker available to use for an exclusion gate. This should leave a population of mostly pure neurons.
We've also found that using a singlet gate first to eliminate dead cells, clustered cells, and or debree helps clean up the downstream gating. This will also help prevent myelin from messing with downstream gating due to its messy scatter and auto-fluorescent properties.
Hope this helps. Feel free to ask me directly more specific questions.
Chandra.
Hello,
an other easy way to get viable cells for flow cytometry is to use a Percoll or Sucrose gradient. A lot of protocols are available online. I used it to purified microglial cells and it worked well.
could one sort E18.5 neurons by FACS for example, and plate them afterwards? are there protocols for gentle FACS that would not kill neurons? thanks, v.
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