I am observing a dose dependent effect on HUVEC. I am loading HUVEC with DAF-FM DA from 1uM to 10 uM in HBSS. The higher the DAF-FM DA concentration, the more cells detach from the plate and are lost during the washing steps.
well, stock is definitely in DMSO (manufacturer suggests). I dilute the probe in HBSS with Ca,Mg. actually i have tested different concentrations of DMSO. up to 1% DMSO, 30 minutes incubation does not have any effect on cells. I had also tried diluting in EGM. none of the cells uptook probe and they were perfectly healthy. then i tested the effect of different buffers PBS, HBSS and media (for comparison). one hours incubation didn't have any noticeable difference.
that's when i tested different concentrations of the probe and saw that dose effect i was talking about. anything above 1uM has detachment effect.
last friday i did an experiment. i loaded the cells with 1uM DAF-FM DA for 1 hour. nice result. very minor cell detachment, cells are in shape, no dead cells and a lot of probe loaded. i was thinking of allowing more loading time (to let more probe to load into the cells). what do you think of that?
I incubate my cells with 1uM DAF-FM in 0.2% FBS phenol red free media for 30 min, and the stain is amazing. If you are afraid to lose fluorescence, I suggest to image your cells (if you are performing IHC analyses) right away or within 24hrs.
i think i am settling for 1uM DAF-FM and 1 hour incubation. i will try to use the PR and FBS free media.
and thank you both for the suggestions. i will keep you posted of my progress
I have used DAF-FM-DA on live embryos and not on cell culture. May be you can reduce the concentration of the reagent and also check that DMSO is not responsible of the effect.
thanks Anna. i have checked for DMSO toxicity. i am going to use 1uM DAF FM till another issue pops up :-)
I have used DAR 4M AM dye for NO detection. There are quite a few issues to optimise for adequate uptake of the marker and then good signal. And I believe it ll depend on the cell types too and it ll vary accordingly. Few things I can suggest are temp at which you load the cells and the temp of the loading solution. Time duration... obviously as you discovered it can really vary (for my experiments it was 25-60 minutes), gentle washing: gentle because sheer forces on the cells by the fluid flow on cells while washing can 'open' up the membrane enough to let the dye leak out so be as gentle as you can be, shelf life of the stock/working solution w.r.t to exposure to light and thaw refreeze cycles, i avoided coloured reagents for the steps from loading to imaging, and finally have a look on caging/uncaging involved for the marker as that affects the final signal too, especially if you are doing quantitative stuff. Patience is the key ingredient with NO detection and quantification :) good luck.
in the last experiment, i took a photo of the cells just before washing the dye off. the cells looked ok. after washing the wells, the cells are all round balls. they are not dead though. i'm not sure if i can be more gentle than i already was. if any of your could share a video of how you washed your cells after loading the dye, it will be really very helpful. just to inform you, i am using a 24-well tissue culture plate from Corning
well, 1 uM DAF-FM DA did not cause cells to detach or die. Any concentration of DAF-FM DA more than that caused cell detachment. the extent of detachment is directly proportional to the concentration of the probe.
however, the cells loaded with i uM DAF-FM DA gave fluorescence signal that decayed over time. bradykinin stimulation could not make any difference in the signal intensity. so i added hydrogen peroxide to check if there is any probe left in the cells. if there is any, then it will readily oxidize and will give a short but intense fluorescence signal. unfortunately, there was no DAF-FM DA left in the cells to react with h2o2.
that's where the twist is. if i load cells with higher DAF-FM DA concentrations, the cells lost integrity. even if the cells show difference in signal upon bradykinin stimulation, the finding is rendered void because the response is not coming from a healthy cell. when 1 uM DAF-FM DA is used, the cells remain healthy, but the signal intensity depletes fast over time and bradykinin stimulation doesn't show any detectable difference in signal intensity
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