Intracellular calcium imaging experiments

Summary

Source : Practical Laboratory Techniques in Neurobiology

Operation method

basic program

Principle

Calcium ion is an important intracellular second messenger involved in many important cellular physiological activities and pathological processes, therefore monitoring changes in intracellular calcium ion levels is important to understand the activity status of cells. Intracellular calcium imaging technique is used to monitor changes in intracellular calcium ion concentration by loading calcium indicator into the cell and utilizing the characteristics of the change in fluorescence intensity or spectral properties that occur when the calcium indicator binds to calcium. Currently, the commonly used calcium indicators are mainly chemical fluorescent indicators, such as Fura-2,Fluo-3, etc.. Fura-2 is a ratio-type indicator, which changes its spectral properties in the calcium free and calcium-bound states, such as in the calcium-bound state, its excitation peak from 363 nm to 335 nm, while the emission peak does not change significantly, so it is usually used to double-wavelength (340 nm and 380 nm) two successive excitation, and the ratio of the emitted light intensity obtained after the excitation is the relative value of the calcium signal (Figure 3-12). 3-12). Since the data of Fura-2 is in the form of ratio, it is not limited by the experimental equipment, indicator loading concentration, cell type and individual experimenter's operation, etc. It can also eliminate the influence of factors such as changes in the thickness of the cell and the redistribution of the indicator in the cell, etc., so that it can truly reflect the changes of the calcium signals in the cell. Therefore, Fura-2 is the most widely used chemical fluorescent calcium indicator.

Materials and Instruments

Neural tissue
Fura-2AM PluronicacidF-127(20%) Ringer's fluid or Artificial Cerebrospinal Fluid (ACSF) 1M HEPES solution Preparation of 1M HEPES stock solution
Fluorescence microscope Monochrome light source system High-speed, high-sensitivity digital cold CCD Microimaging software Other Fura-2-specific filter sets

Move

1. Cultured cells for calcium imaging:

(1)Specimen preparationRats or mice were killed by cervical dislocation or hypoxia, and the cells were taken, separated and dispersed then cultured for 3-7d.

(2) Fura-2AM solution preparation Fura-2AM was first dissolved in DMSO to prepare a 1OmM stock solution, and then split the device at -20°C. 1OmM of Fura-2AM was dissolved in Ringer's solution with the co-diffusor PluronicacidF-127 (20%) during the same day experiments, as formulated in Table 3-l.

(3) iura-2AM-loaded cells The above configured Fura-2AM solution was added to the cultured cells (24-well plate) and incubated at room temperature for 30 min, after which the Fura-2AM solution was aspirated and added to the normal Ringer's solution for elution for 30 min, and then left to be used. During the whole process, the 24-well plate was wrapped with tin foil and protected from light.

(4) Calcium imaging of cultured cells was performed by placing Fura-2-loaded cells on a fluorescence microscope carrier stage, and successively applying the excitation wavelengths of 340 nm and 380 nm to each exposure for 30 s, with an exposure frequency of 1 Hz, and calculating the F340/F380 ratio to represent the relative calcium level. The F340/F380 ratio was calculated to represent the relative calcium level. When a certain agonist that can induce an increase in intracellular calcium was given by perfusion, a change in fluorescence intensity could be observed, which was characterized by an increase in fluorescence intensity at the excitation wavelength of 340 nm and a decrease in fluorescence intensity at the excitation wavelength of 380 nm, and the F340/F380 ratio was therefore increased -.

2.Acute tissue thin-section calcium imaging;

(I) Specimen preparation After rats or mice were anesthetized by intraperitoneal injection of uracil, the material was taken, and 350-450 µm thick tissue slices (spinal cord slices or brain slices) were cut on a vibrating slicer and incubated in artificial cerebrospinal fluid passed through a mixture of 95% 02 and 5% C02.

(2) Fura-2AM (1OµM) solution was prepared as above by first dissolving Fura-2AM in DMSO to prepare a 1OmM stock solution, and then dispensing the device at -20°C. 1OmM of Fura-2AM was dissolved in artificial cerebrospinal fluid (ACSF) with the co-diffusor PluronicacidF-127 (20%) at the time of the day's experiment, as formulated in Table 3-2.

(3) Fura-2AM loaded cells Tissue slices (spinal cord slices or brain slices) were placed in the above formulated Fura-2 solution and incubated for 45 min. After loading was completed, they were placed in normal artificial cerebrospinal fluid free of Fura-2 for 30 min for elution, and then calcium determination was started. Note that a gas mixture of 95% 02 and 5% CO2 was continuously vented throughout the process, and it was performed under light protection.

(4) Calcium imaging of tissue slices (spinal cord slices or brain slices) after Fura-2 loading was placed on the fluorescence microscope carrier stage, and the excitation wavelengths of 340 nm and 380 nm were successively applied to expose the tissue slices for 30 ms each at an exposure frequency of 1 Hz, and the F340/F380 ratio was calculated to represent the relative calcium level. As shown in Figure 3-14 on the color page, the calcium level of the cells in the superficial region of the dorsal horn of the spinal cord was induced to increase significantly after electrical stimulation of the dorsal root, which was manifested by the increase of the fluorescence intensity at the excitation wavelength of 340 nm and the decrease of the fluorescence intensity at the excitation wavelength of 380 nm, and thus the F340/F380 ratio was increased.

Caveat

1. To avoid or minimize fluorescence quenching, all operating procedures should be carried out under light protection.

2. Since Fura-2AM produces acid during degreasing, HEPES needs to be added to the solution to maintain pH stability. Otherwise, excess acid will lead to cell death.

Common Problems

1 Observe the state of the cells or tissue sheets before Fura-2AM loading, the state of which determines the efficiency of Fura-2AM loading. Temperature has no absolute effect on the efficiency of Fura-2AM loading, and loading at room temperature or 33°C-37°C is acceptable.The time of Fura-2AM loading cells should be controlled at 30-60 min. The addition of HEPES during the configuration of Fura-2AM solution is especially important because Fura-2AM produces acid during the process of crossing the cell membrane to be sheared by esterase to form Fura-2, and therefore HEPES needs to be added to the solution to maintain the stability of pH. Otherwise, excess acid will lead to cell death. When applying 340nm and 380nm excitation wavelengths to excite fluorescence successively, the exposure time is generally 10-50ms, and too long an exposure time will easily cause fluorescence quenching. During calcium indicator loading of spinal cord slices or brain slices, it is desirable to continuously pass a gas mixture to ensure a good cellular state.

2 In addition to the above method of loading the esterified calcium indicator Fura-2 into cells by incubation for calcium determination, it is also possible to apply a microelectrode to load the calcium indicator directly into the cells. This method is the loading method commonly used for calcium determination in conjunction with electrophysiological membrane clamp technology. The indicator is dissolved in the intra-electrode solution, and after forming a whole-cell recording of the cell, the indicator passively diffuses into the cell through the low-impedance electrode tip. Loading of the indicator using this method can be done for any of the indicator forms. After the indicator is loaded into the cell, the calcium determination procedure is as described above.

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