Summary

Calcium ions play an important role in many physiological activities. Cytofluorescence calcium ion imaging is an imaging technique that utilizes calcium ion indicators to convert changes in intracellular calcium ion concentration into fluorescent signals, thereby converting cellular electrical activity into recordable optical signals.

The activity of intestinal glial cells is closely related to calcium ions, and the use of calcium ion indicators to monitor the activity of glial cells is particularly important. Calcium ion fluorescent indicators are almost non-fluorescent before binding calcium ions, and the fluorescence intensity is significantly enhanced after binding with calcium ions.

Operation method

Fluo-4 AM Fluorescence Calcium Ion Imaging

Principle

Calcium ions play an important role in many physiological activities. Cytofluorescence calcium ion imaging is an imaging technique that utilizes calcium ion indicators to convert changes in intracellular calcium ion concentration into fluorescent signals, thereby converting cellular electrical activity into recordable optical signals. The activity of intestinal glial cells is closely related to calcium ions, and the use of calcium ion indicators to monitor the activity of glial cells is particularly important. Calcium ion fluorescent indicators are nearly non-fluorescent before binding calcium ions, and the fluorescence intensity is significantly enhanced after binding calcium ions. We used Fluo-4 AM fluorescent calcium ion probe as an example to observe the excitatory changes of enteric glial cells.

Materials and Instruments

Sterile PBS solution, 5 μM Fluo-4 AM, DMEM/F12 medium
Confocal Petri dish, sterile tip
Turntable confocal microscope

Move

(1) Inoculate primary enteric glial cells on confocal culture dishes one day in advance;

(2) After observing the cells in good condition on the next day, the culture medium was discarded and PBS washed three times;

(3) Add appropriate amount of DMEM/F12 medium containing 5 μM Fluo-4 AM (Beyotime, S1060) and incubate at 37 ℃ for 30 min.

(4) PBS was washed, placed on a turntable confocal microscope (UltraVIEW VoX, PerkinElmer) for observation (imaging for 30-60 s to collect basal state images, add the appropriate drugs and collect the images), and used Volocity Demo 6.0 (PerkinElmer Inc.) for ^Ca2+ analysis.

Caveat

Prepare the cells one day in advance, the amount of cells is moderate, not too much, not too little, to ensure that there is a slight gap between the cells in the high magnification field of view but not excessively loose;Wash the cells gently, the tip of the gun should not directly touch the cells, and the liquid should flow down slowly along the wall of the petri dish;The probe is fully incubated and protected from light;Put on the turntable confocal microscope to observe the dosing, try to give the drug at the edge, in order to prevent the drug delivery operation affect the cell state;.After the probe incubation, wash the probe well to eliminate the interference of residual probe;If the fluorescence intensity is too weak, the incubation time can be extended or the probe concentration can be increased appropriately;

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