What is your favorite method of quantifying DNA release of neutrophils? Is anybody using a plate reader approach? I have read about counting cells in fluorescence images but this might not only be tedious but also not always very exact or you'll need to have software that calculates the fluorescence intensity of the spots (but maybe someone knows a nice imageJ macro?).
I have tried DAPI and plate reader in 24° well plates and it worked some but maybe I overlooked why I could not use this method in general. I know about picogreen but its quite pricey.
I also know that DAPI also enters living cells and that it also binds RNA etc pp, but maybe its a question of timing and precision?
I was measuring DNA released from stimulated neutrophils by:
1) treating the cells with 1U/ml of MNase 15min. at 37C.
2) stop the reaction by adding EDTA 2-5mM (depending of your calcium concentrartion in your medium)
3) Take the supernatant and stain your DNA with a fluorochrome, like picogreen but i think it can also work with DAPI.
I was reading in a fluoroscan which can read 96 well-plates.
Good luck
We have developed an imaging system where you can count automatically DAPI stained cells. I think it should work for activated polymorphs as well. In case you are interested please contact Dr. Peter Schierack at the Lausitz University: 03573 85932.
We use the technolgy for double strand break detection in chromatin (Runge et al., Int J Radiat Biol, 2012;88(5):439-447)
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