I am trying to measure change in cellular calcium ion concentration using aequorin expressing plant. I could find many research article and protocols about the measurement using the same system, so I summarized and followed them. However, the signal seems so weak even when I applied twice as much amount of coelenterazin as previous reports. It required at least 30s exposure for our instrument, therefore, not applicable for the purpose of my study. The 30 seconds exposure still does not show any distinctive difference between non-expressing samples and aequorin expressing samples. Even after discharge with CaCl2 and methanol, the signal doesn’t get any stronger. The instrument is NightShade, and sensitivity of the instrument seems fine, based on other luminescence measurement such as luciferase expressing sample treated with luciferin.
If you have an experience in this experiment, please let me know how to solve the problem.
Thanks in advance.
The best way to measure intracellular calcium is imaging with indicators like Fura-2, Fluo-3, Fluo-4, etc. The signal to noise ratio is usually quite high. Another approach is to use genetically encoded calcium indicators (GCAMPs), See: Monitoring activity in neural circuits with genetically encoded indicators. Broussard GJ, Liang R, Tian L. Front Mol Neurosci. 2014;7:97
.
The best way to measure intracellular calcium is imaging with indicators like Fura-2, Fluo-3, Fluo-4, etc. The signal to noise ratio is usually quite high. Another approach is to use genetically encoded calcium indicators (GCAMPs), See: Monitoring activity in neural circuits with genetically encoded indicators. Broussard GJ, Liang R, Tian L. Front Mol Neurosci. 2014;7:97
.
The signal from the aequorin happens very fast. are you injecting your sample and then measuring?
Fura-2 is the most frequently used fluorescent dye for measuring intracellular calcium.
Grynkiewicz G, Poenie GM, Tsien RY. A new generation of Ca2+ indicators
with greatly improved fluorescence properties. J Biol Chem 1985; 260:
3440–3450.
Soldati L, Adamo D, Zerbi S et al. Erythrocyte voltage-dependent calcium
influx is reduced in hemodialysis patients. Kidney Int 1999; 56: 190–197.
Hi Seunghye,
I wouldn't recommend using a bioluminescence approach as the number of photons released during bioluminescent reactions is many orders of magnitude lower than those released during fluorescence reactions per unit time.
As Menachem and Giuseppe mentioned, fluorescent calcium indicators come as organic dyes (like Fura-2) or genetically encodable proteins (like the GCaMPs and Cameleons). Whichever route you choose, if you have the ability to use a radiometric indicator (including Fura-2 and the Cameleons) using such an indicator will greatly facilitate your ability to determine absolute calcium concentration.
Amy Palmer and Roger Tsien put out an excellent step by step methods paper for finding Ca2+ concentrations based on fluorescence output of radiometric sensors. I'd recommend using it as an in depth guide whichever route you choose:
Amy E. Palmer and Roger Y. Tsien 2006 Measuring calcium signaling using genetically targetable fluorescent indicators. Nature Protocols, Vol 1: 1057-1065.
Best of luck,
Joey
Hi Seunghye,
I suggest that you used patch clamp recorder for measuring changes in cellular Ca2+ concentration invitro cell
Bets regard
Dear researchers,
Thank you all for all the comments. Your comments are really helpful.
Thanks.
Best wishes,
Seunghye
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