Dear all,
I'm trying to understand if a molecule of my interest (capable of inducing glucose-stimulated insulin secretion) determines an increase in intracellular calcium levels in the presence of high glucose concentration (25 mM) in INS-1E cells.
To this aim, I'm using Fluo 3-AM molecuar probe.
In particular:
- INS-1E are seeded and grown in 96-well (clear bottom, black walls) for 24 h in growth medium.
- Cells are then cultured in 3 mM glucose KREBS Buffer for 1 hours.
- Afterward, cells are cultured with 5 µM Fluo 3-AM dissolved in 3 mM glucose KREBS Buffer + 0.02% Pluronic for 1 h.
- After 3 washes with PBS, cells are cultured again in 3 mM glucose KREBS Buffer for further 30 minutes, to allow the de-esterification of the probe.
- Depending on the condition, the cells were kept in 3 mM glucose KREBS Buffer or stimulated with 25 mM glucose KREBS Buffer, or 40 mM KCl (positive control).
Under these conditions I'have verified the entry of the Fluo 3-AM in the cells by fluorescence microscope: cells without the probe (blank) showed no signal, while cells with the probe were fluorescent (NB: the fluorescent signal decayed very quickly).
Then, the signal was read by a fluorometer with the following setting:
- reading from the top
- excitaiton flter: 485 nm
- emission filter: 535 nm
- counting time: 0.3 s
- lamp power: 10320
I report below the fluorescence values obtained:
- Blank: 7785
- 3 mM glucose KREBS Buffer: 8351
- 25 mM glucose KREBS Buffer: 8317
- 40 mM KCl: 8469
Is it normal for blank to have such high values?
High glucose and KCl do not increase fluorescence. How is it possible?
Do you have any suggestions?
Thank you very much
Nicola
Ciao Nicola,
maybe I didn't understand everything ...
How is the optical measuring setup? Is the excitation from below?
Have you checked the pH's and is the ionic strength of solutions almost the same?
My understanding is that your Krebs-buffer is without calcium chloride
and sodium bicarbonate....
What is the meaning of WHITE in "Is it normal for white to have such high values?"
Ti auguro il meglio.
Saluti
Tobias
Dear Tobias,
Excitation is from the top.
This is the KREBS buffer composition: 10 mM HEPES, 114 mM NaCl, 4.4 mM KCl, 1.28 mM CaCl2, 1 mM MgSO4, 29.5 mM NaHCO3; pH 7.4 (with the addition of 0.1% BSA and 3 mM or 25 mM glucose).
These are the same solutions I use for the evaluation of glucose-stimulated insulin secretion....
"White" means "blank" (sorry for the inaccurancy).
Thank you
Nicola
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