I am trying to do immunocytochemistry to characterize the cells in a culture. As I am actually differentiating a type of neuron from neural stem cells, and I have done once the differentiation and ICC using 2 different markers. My question is, as not all neural stem cells differentiate and some of them remain as normal neurons (I have used MAP2, TuJ1 to stain the normal neurons), so how to calculate exactly the population of each cell type in a culture, as I would need to study the changes in the neuron of interest only. What methods should I use? I don't think qPCR is a good method as it only gives a precise quantitation of markers express from the culture but not the their populations. Please give me some advice. Thank you.
Hi,
I am not pretty sure if I understand right your question but I think that if you take photos of your ICCs, you will be able to know your cell population. Using confocal microscopy brings you valuable information about ICC and allows you precise quantification.
Viewing cells with DAPI channel, you'll know total amount of cell population (you can count how much cells you have in an area). Then, MAP2 and Tuj1 positive cells can be counted and tagged as "normal neurons" and negative ones (only with DAPI stain) will be neural stem cells. As a suggestion I recommend you to use an antibody against Nestin or SOX-2 to stain neural stem cells. Results can be viewed as percentage of cells MAP2/Tuj1 positive +/- standard error.
Hope it helps.
It has been a while since I have done this, but if you have replicate plates, some of which are expendable, you might try flow cytometry. I hope that you can get help from someone that already has a flow cytometer.
Single cell transcriptomics is of course the best option for defining cell types. Any histochemistry is gong to require significant troubleshooting per label.
To answer your question, you can make progress using cellProfiler, but like the other answers you will need a marker to identify all cells. They mention DAPI (i'd add hoeschtt 33342), this can work very well, but i prefer a label for the cytoplasm of the cells.
Another thing to keep in mind, you will have easier time working with a sparse culture. And i'm always believed a larger field of view is best
Good Luck!
you can do ICC with 2-3 antibodies + dapi, and quantify the results (best if you do it in triplicate). You can also do flow cytometry, which is very informative, if you can afford to spare ~1 million cells/each staining experiments. You can combine ICC and flow result, which is the best. For example, see one of our older papers PMID: 19609935
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