Hi everyone!
I want to identify by flow cytometry the immune cells found in the brain with a localized injury.
My hypotesis is that the injury will attract more/different immune cells respect to the healthy tissue. If the immune cells are "attracted" by the lesion, I expect a gradient with more immune cells close to the injuried spot and less moving far from it.
Now, I see a problem in the harvesting process: if I collect a small area around the lesion I'll find a higher % of immune cells but if I harvest a bigger area, I'll find a smaller %, just because of this "gradient".
To be consistent between different mice I should harvest the same-sized area around the lesion, which I don't see practically possible.
How should I do to be able to compare between different brains?
Harvesting the entire brain or at least the entire hemisphere to have a consistent sample size?
Is this problem of "non-uniform" tissue relevant also in comparing cell subsets? For example comparing the different subpopulations of CD45+ cells
Thank you very much for the help.
Filippo
Hello Filippo,
I see 2 possible ways of going at this.
1) Establish multiple areas of interest within the injured hemisphere. For example
A. Injured tissue
B. Tissue right next to the lesion (transition area)
C. Remote area
or whatever fits your experiment. Such experiments are used for other animal models (of induced AMI for example) as it was shown that these 3 areas have different immune cell compositions. Also, these are the 3 main distinct areas of interest in the brain tissue of patients with ischaemic stroke.
2) If it's easier for you and it fits your experiment, you can just analyse the injured hemisphere as a whole and use the uninjured hemisphere as control.
The first (1) way, i think, is better at showing the spatial distribution of immune cells and provides a better understanding of how cells migrate to the lesion... But it is more complex and elaborate and, as you said, harvesting tissues of equal size will be problematic between animals.
The second (2) way is simpler, but you will have less resolution as the immune cells you will analyse will be a mixture of cells from all 3 regions. However, in this case, you can "normalize" your data between animals by introducing a cellularity index - ratio between % of desired cells from total immune cells in the injured hemisphere, and % of desired cells from total immune cella in the control hemisphere. As long as the lesion is approximately equally sized between animals, this index would be a better indicator. If the lesions vary in size, you can add a correction factor such as lesion area or whatever.
Better off, try to read some papers that adress similar issues. Maybe some experimental models of ischaemic stroke in rodents could help you establish your protocol?!
I think u should add IHC to your methodology. U can characterize the cells by FCM and then drive in your point of higher number of immune cells near the lesion by IHC.
I will also suggest using chemokine markers to show unregulated recruitement of immune cells.
As suggested previously, its a good idea to use the healthy hemisphere as your control.
As for regulating the amount of tissue u harvest, u can use punch biopsies or harvest tissues of equal weight. However, i don't see how you might get the same size or location of lesion in the mice brain as that might be a confounding factor for immune phenotype.
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