Calcium Green 1AM

Protocol (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock Solution Preparation
Solvent: DMSO

2. Working Solution Preparation
2.1 Working concentration selection:
- Calcium Green 1AM 4 mM.
- Calcium Green 1AM 10 mM, dissolved in oxygenated artificial cerebrospinal fluid (a-CSF).

2.2 a-CSF composition for 10 mM working solution: 120 mM NaCl, 8 mM KCl, 1.26 mM CaCl2, 1.5 mM MgCl2, 21 mM NaHCO3, 0.5 mM Na2HPO4, 30 mM glucose, pH 7.4.

2.3 Note: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For primary hypothalamic astrocytes passaged onto poly-D-lysine-coated glass coverslips[1]:
- Load cells with 4 mM Calcium Green 1AM and incubate at 37°C for 30 min.
- Place the coverslip in a temperature-controlled perfusion chamber on the stage of an inverted microscope.
- Perfuse with oxygenated Kreb's solution at a rate of 1.0 mL/min.

3.2 For isolated embryonic mouse hindbrain preparations[2]:
- Use isolated brainstem for embryonic parafacial oscillator (e-pF) experiments and transverse medullary slices for preBötzinger complex (preBötC) experiments.
- For e-pF imaging, place the isolated brainstem ventral side (pial side) up.
- For preBötC imaging, place the transverse slice rostral side up.
- Incubate the preparation in oxygenated a-CSF containing 10 mM Calcium Green 1AM for 40 min.
- After loading, transfer the preparation to the recording chamber and continuously perfuse with oxygenated a-CSF at 30°C.
- Place the preparation in the recording chamber and recover in oxygenated a-CSF for 30 min to wash out excess dye.
- Allow spontaneous firing activity to appear during this recovery period.

4. Controls
4.1 Negative control: Cells on control coverslips perfused with Kreb's solution alone show no change in calcium signal.

4.2 Positive control: ATP (50 mM) applied for 30 s reliably evokes calcium oscillations and, at the end of the study, induces a strong and sustained increase in calcium response on each coverslip.

4.3 Specificity control: No calcium influx is observed in cells perfused with heat-inactivated leptin or chemical inhibitors of mitogen-activated protein kinases (MAPK), indicating specificity.

5. Detection and Analysis
5.1 Instrument type:
- Inverted microscope.
- Epifluorescence microscope equipped with a cooled CCD camera.

5.2 Wavelength settings:
- Calcium Green is excited by an argon laser, and emission is captured at 488 nm.
- Use a fluorescein filter block to excite the dye and capture emission.

5.3 Imaging parameters:
- Perform real-time imaging using a 40× objective at a frequency of 2 frames/s, capturing 600 frames in total.
- Capture images in overlapping mode with 100 ms exposure during the 30 to 120 sec time period.

5.4 Result analysis:
- Leptin (6.2 nM) induces a strong increase in intracellular Calcium Green fluorescence in nearly 100% of astrocytes; the response appears immediately after the start of 2 min perfusion, rapidly returns to baseline, with an average increase of 22.7 ± 3.3% (n=20).
- Repeated application of leptin on the same coverslip after 5 min induces a much smaller response amplitude (4.5 ± 1.1%), indicating desensitization.
- Calculate the percentage of responsive cells and the magnitude of increase relative to baseline fluorescence intensity for each coverslip.
- All results are repeated at least four times.
- Calculate the average intensity within the region of interest for each frame.
- Measure the average baseline fluorescence between two spontaneous events, normalize the DF signal, and display as ΔF/F.
- Analyze cells that take up the dye and can be detected using the imaging parameters used as active cells.
- Expected results: Rhythmic organized calcium transients are detected in active cells, manifested as fluorescence changes.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.