DNase and TdT
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Tue, 16 Jul 1996 08:47:12 -0500
DNase-treated cells are not a good positive control for DNA strand
break labeling assays. During cells exposure to DNase there is no
control, whatsoever, as to how much DNA is degraded and how much
leaks out from the cell during the incubation and afterwards, during
the strand break labeling reaction, which has many centrifugations.
We found that HL-60 cells (other myelocytic cell lines can be used as
well) incubated with 0.15 uM camptothecin for 3 to 4 h offer an
excellent and reproducible control. During the incubation nearly all
S phase cells become apoptotic, having extensive DNA breakage. In
contrast, G1 and G2/M cells have no DNA breaks, and serve, in the
same sample, as negative control. If one prepares a large batch of so
treated cells, they can be used stored in fixative and used for many
months. Some vendors (Phoenix Flow Systems) sell such control cells.
The critical issue in inducing apoptosis of S phase cells is that the
cells have to move through S phase rapidly. If cultures are somewhat
subconfluent (e.g. at densities over 6 x 105E) the rate of DNA
replication drops and the S phase become more resistant to
camtpothecin.
By the way, if one is using the nuclease treated cells, it may be
better to use micrococcal nuclease, which, similar to the
apoptosis-associated nuclease(s) has preference to the
internucleosomal (linker) DNA sections, rather than DNase.
Zbigniew Darzynkiewicz
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