Application Data (Detecting Response of Intracellular Ca2+)

Application Data

- Detecting Response of Intracellular Ca²⁺ -

Feature

Feature Ca²⁺ existing inside cell is one of messengers who transfer external information into cell.
As an example, Concentration of Ca²⁺ in cell raises when the cell is in state of excitation.
Measurement of intracellular Ca²⁺ can be performed by instrument such as microscopy and spectrofluorometer with fluorescence indicator such as Fura2 and Fulo 3. In case the measurement is performed with microplate reader, throughput can be higher than it is done by other kind of instrument.
Fura2 is being combined with Acetoxymethyl AM Ester/AM when it is outside cell. When the Fura2-AM enter into cell, it its de-esterified by enzyme Esterase. Furas2 which is detached from AM would combine with Ca²⁺, and would become fluorescent depending on concentration of Ca²⁺.
MTP-800AFC Microplate Reader makes Ca²⁺ measurement of multiple sample at short time thanks to its dual-Ex/Dual-Em measuring function.


Load test on cell with FL indicator	:	1sample/hour
On microplate	:	10 samples /hour

Ca Measurement of Fura 2-Sensitive FL indicator

Procedure of Analysis

(Fura 2 Meas. Flow diagram)

Incubation on Microplate
(HeLa Cell 3to 5days)

Loading agent
(Fura 2-AM 10µM, 30min)

Dual W.L.(340/380nm)
Excitation

Detection of FL(510nm).

Ratio calculation(EM/EX)

* Ionomycin shall be dispensed one minute after
starting measurement of emission light.

Reagent

Fura 2-AM special packaging
<Price> 18,200yen/JPN (for 20 plates)
<Manufacturer> DOJINDO <Distribution> Wako

Instrument

MTP-800AFC	4,400,000 yen
Filter: 340nm(EX)	77,000 yen
Filter: 380nm(EX)	77,000 yen
Filter: 510nm(EM)	46,000 yen
DP-50 Dispenser	600,000 yen
Intercellular Ca unit	850,000 yen
Optical plate shall be chosen.

This experiment is performed under support of Dr. Sakurai at Hamamatsu Medical School Photon Medical Research Centre.

You can monitor Ca-response being induced by dispense of reagent