A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Summary

Due to the spectral overlapping of the excitation and emission wavelengths of NADH and fura-2 analogs, the signal interference from both chemicals in live cells is unavoidable during quantitative measurement of [Ca²⁺]. Thus, a novel online correction method of NADH signal interference to measure [Ca²⁺] was developed.

Abstract

To measure [Ca²⁺] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in live cells because of autofluorescence interference, mainly from nicotinamide adenine dinucleotide (NADH). More specifically, this is a major obstacle when measuring the mitochondrial [Ca²⁺] quantitatively using fura-2 analogs because the majority of NADH is in the mitochondria. If the fluorescent dye concentration is the same, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Based on this principle, a novel online correction method of NADH signal interference to measure [Ca²⁺] was developed, and the real signal intensity of NADH and fura-2 can be obtained. Further, a novel equation to calculate [Ca²⁺] was developed with isosbestic excitation or excitation at 400 nm. With this method, changes in mitochondrial [Ca²⁺] could be successfully measured. In addition, with a different set of the excitation and emission wavelengths, multiple parameters, including NADH, [Ca²⁺], and pH or mitochondrial membrane potential (Ψ_m), could be simultaneously measured. Mitochondrial [Ca²⁺] and Ψ_m or pH were measured using fura-2-FF and tetramethylrhodamine ethyl ester (TMRE) or carboxy-seminaphtorhodafluor-1 (carboxy-SNARF-1).

Introduction

The significant role of intracellular Ca²⁺ is widely known¹. The quantification of [Ca²⁺] is essential to understand the processes of the cellular physiological functions. Fura-2 analogs are quite useful because they are excited in the UV range (<400 nm), and the ratiometric method can be applied for the quantitative measurement. Therefore, other physiological parameters such as pH, membrane potential, etc., can be measured with other fluorescent dyes. The mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) range was reportedly 0.08−20 μM^2,

Protocol

All experimental protocols were approved by the local institutional animal care and use committee.

1. Solution preparation

1. Prepare single freshly isolated cardiac myocytes¹¹.
   NOTE: Each laboratory might have a different cell storage solution. Here, the myocytes are stored in culture medium (DMEM).
2. Prepare 100 mL of Ca²⁺-free solution (Table 1).
3. Prepare 50 mL of culture medium in a 50 mL beaker. Aliquot 5 mL and put it in a water bath at 37 °C. Keep the remaining solution at room temperature.
4. Prepare 50 mL of the saponin solution by addi....

Results

Mitochondrial Ca²⁺ changes due to correction¹⁰
Figure 4 shows the changes in [Ca²⁺]_m before and after the correction. The results clearly showed the substantial changes in [Ca²⁺]_m. The mitochondrial resting calcium concentration without cytosolic Ca²⁺ ([Ca²⁺]_c) was 1.03 ± 0.13 µM (mean ± S.E., n = 32), and the maximum [Ca²⁺]_m at 1-µM [Ca²⁺]_c was 2.......

Discussion

The interference correction method was successfully developed for measuring the signals of NADH and fura-2 analogs. Exact measurement of the signals is essential for exact correction. However, the inherent nature of the fluorescent device produces a background signal unrelated to that of NADH of fura-2. The highest quality band-pass filter can only pass up to 10⁻⁸ of the unwanted wavelengths of the light. However, the fluorescent signal from a single cell is very small, and the reflection of the excitati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mL eppendorf tube	Axygen	MCT-200-C	2 mL Tube
AD/DA converter	Instrutech	ITC-18	Equipment
ADP, Adenosine 5′-diphosphate monopotassium salt dihydrate	Sigma-aldrich	A5285	Chemicals
Band pass filter	Ealing Electro-Optics, Inc	35-3920	Equipment, 640±11nm
Band pass filter	Omega Optical	690-9823	Equipment, 590±15nm
Band pass filter	Omega Optical	500DF20-9916	Equipment, 500±20nm
Band pass filter	Chroma Technology Corp.	60685	Equipment, 450±30nm
Calcium chloride solution	Sigma-aldrich	21114	Chemicals
carboxy-SNARF-1(AM)	Invitrogen	C1272	Chemicals
Charge-coupled device (CCD) camera	Philips	FTM1800NH/HGI	Equipment
Dichroic mirror	Chroma Technology Corp.	86009	Equipment, Multiband dichroic mirror, Reflection : <400nm, 490±10, 560±10, Transmission : 460±15, 510±20, >580nm
Dichroic mirror	Chroma Technology Corp.	567DCXRU	Equipment, Reflection : <560nm, Transmission : > 580 nm
Dichroic mirror	Chroma Technology Corp.	480dclp	Equipment, Reflection : <470nm, Transmission : > 490 nm
Dichroic mirror	Chroma Technology Corp.	20728	Equipment, Multiband dichroic mirror, Reflection : <405nm, 470±30, Transmission : 430nm~520nm, > 640 nm
Dimethyl sulfoxide(DMSO)	Sigma-aldrich	154938	Chemicals
DMEM, Dulbecco’s Modified Eagle’s Medium	Sigma-aldrich	D5030	Chemicals
EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid	Sigma-aldrich	E4378	Chemicals
FCCP, Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone	Sigma-aldrich	21857	Chemicals
field diaphragm	Nikon	86506	Equipment
Fura-2-FF(AM)	TEFLABS	137	chemicals
Green tube	DWM		test tube
HEPES,  4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)	Sigma-aldrich	H3375	Chemicals
High-speed counter	National Instruments	NI-6022	Equipment
Hot mirror	Chroma Technology Corp.	21002	Equipment, 50:50
Inverted microscope	Nikon	TE-300	Equipment
Malate	Sigma-aldrich	27606	Chemicals
Near infrared filter	Chroma Technology Corp.	D750/100X	Equipment, 750±100nm
Oil immersion lens	Nikon	MRF01400	40x, NA 1.3; Equipment
Photon counter unit	Hamamatsu	C3866	Equipment
Photon multiplier tube	Hamamatsu	R2949	Equipment
Polychrome II	Till Photonics	SA3/MG04	Equipment
Potassium chloride	Merck	1.04936	Chemicals
Potassium hydroxide solution	Sigma-aldrich	P4494	Chemicals
Pyruvate	Sigma-aldrich	107360	Chemicals
Rotenone	Sigma-aldrich	R8875	Chemicals
Saponin	Sigma-aldrich	S4521	Chemicals
TMRE, Tetramethylrhodamine, ethyl ester	Molecular probes	T669	Chemicals