Who has expeience using Hoechst uptake as an indicator of apoptosis?
1) Are you using 33342 or 33258? Concentration?
2) Are you using log or lin acquisition for the Hoechst?
3) Do you use PI exclusion? How about 7-AAD? Concentration?
4) How long do you expose the cells to the dyes before reading? Temp?
My concerns mostly center around the third question... the 7-AAD signal
just doen't look like I expect it should. Several labs I work with have
been using the following protocol with various cell types, including
chondrocytes, HL-60, and fresh human T cells:
1.Resuspend at least 0.5 x 10^6 cells in 12x75mm tubes at a
concentration of 10^6 cells/ml.
2.Add Ho 33258 at a final concentration of 1ug/ml to samples.
3.Incubate the samples in a 37 C waterbath for 7 minutes.
4.Remove samples from the waterbath and immediately place on ice.
5.Add 7-AAD at a final concentration of 1ug/ml to samples.
6.Incubate on ice for 10 minutes.
7.Analyze samples, with log amplification for both Hoechst and 7-AAD.
I almost never see any Hoechst+/7-AAD- cells, no matter what the
treatment. Only double negatives and double positives. If I do an
Annexin/7-AAD expeiment, I see Annexin+/7-AAD- cells, suggesting cells
that are clearly apoptotic, with intact membranes. Aren't these the
type of cell that would accumulate Hoechst but exclude 7-AAD?
One thing to consider is that I am reading 7-AAD off of two differnet
PMTs, and exciting with two different lasers on our FACStar+. I get
good sensitivity & low CVs when lloking at alignment beads in either
configuration, but I still suspect the different 7-AAD staining profiles
may be related to the different configurations.
For Annexin experiments, all excitation is off of the front I90, in
488nm/250mW. Annexin is measured on FL-1 with a 530BP and a 560DS, and
7-AAD is being measured on FL3-1 with a 660BP and the 610DS split
mirror.
For Hoechst experiments, scatter and Hoechst are being excited by the
front I90 in UV/50mW, and 7-AAD is being excited by the rear I70 in
488nm/250mW. Hoechst is being measured on FL1 with a 424BP and a 505DS,
and 7-AAD is being measured on FL4 with a 660BP, 640DL, and the 610DS
split mirror.
Any feedback would be greatly appreciated.
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