I was stained cultured keratinocytes with 75nM Lyso tracker green DND-26 for 30 min, then changed dye-free medium and photoed the cells every 1 minute with time-lapse micrpscopy, total 1 hours.
However, during photoed progress I found the cells (cultured under normal condition without any other stimulation) in DIC chanel had "cell blebbing" phenomenon (since 10 minutes).
Does anyone have some tips about using Lyso tracker and avoiding cell blebbing?
Thanks in advance.
DUERNA
Hi, Wee.
Without lyso tracker, there were indeed had cell blebbing under the normal cultured conition (autophagy?), I once observed the living cells with phase-contrast microscopy, while the blebbing cells were very few.
With lyso tracker, the cells on my observation filed, all the cells get blebbing.
DUERNA
The blebbing means your cells are under stress and they are probably dying. You can use some good anti-oxidants with the lytotracker. You can decrease the fluorescence illumination to reduce photodamage. Lysosomes may release all sorts of toxic digestive enzymes adding to your problem.
Also reduce the time of exposure if you can.
Regards
Hi there
Make sure you optimize the cell number with the concentration of dye. Try for control with more cell number and less concentrated dye for 10 min. good luck
Rahman
@Nurul Kabir
Hi, thank you for your suggestion, it's helpful.
Photodamage seems to be the most possible reason in my situation.
I have reduced the fluorescence illumination, and it workd well.
As for "reduce the time of exposure", I modified my experiment schedule, I will give-up long time observation and focous on one-time point observation.
Thank you again.
DUERNA
Hey Dueerna Tie Sodium pyruvate reduces photo toxicity are supplementing your cells with it?
@Baskar Jesudasan Sam Joshva
Thank you for your suggestion.
I did not know sodium pyruvate could protect cells from phototoxicity, so I serached some information from internet (Celprogen company website):
- "Sodium pyruvate may also help reduced fluorescent light-induced phototoxicity (Spierenberg, G.T., et al. 1984) Phototoxicity of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid-buffered culture media for human leukemic cell lines.( Cancer Research 44:2253)".
Thank you and if I have chance I would like to try sodium pyruvate.
DUERNA
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