If you have a cell culture of the cells, you apply ratiometric dyes like Fura-2 AM up to 2-3 uM free Ca2+-concentrations or Fura-FF AM up to 20-50 uM. There are numerous protocols available, briefly, you pretreat the cells in serum free buffer like HBSS with 2-5 uM of the dyes for 30-40 min, then wash the cells and ten apply alternating excitation of 340 nm and 380 nm with emission detection at 510 nm. From the ratio of the emission of the two excitations the free Ca2+ concentration in the cytoplasm can be calculated. See examples in the link below. Hope it helps, Karoly

https://www.thermofisher.com/hu/en/home/life-science/cell-analysis/cell-viability-and-regulation/ion-indicators/calcium-indicators.html?ef_id=CjwKCAjw3POhBhBQEiwAqTCuBnP8G0Owfwmcslw3Fod3ImgJILc5ILV9LHed14S2UFOk5BB0-zfvFhoCiIQQAvD_BwE:G:s&s_kwcid=AL!3652!3!651042894850!p!!g!!fura-2!2031782395!77553661339&cid=bid_pca_iva_r01_co_cp1359_pjt0000_bid00000_0se_gaw_nt_pur_con&gclid=CjwKCAjw3POhBhBQEiwAqTCuBnP8G0Owfwmcslw3Fod3ImgJILc5ILV9LHed14S2UFOk5BB0-zfvFhoCiIQQAvD_BwE

Hi Fariba, Measurement of [Ca2+]i was done in a single cell preloaded with fluorescent Ca indicator fura2 using a fluorescent unit (lonoquant) consisting of an inverted microscope (Zeiss IM 35) equipped with a mercury lamp and a rotating filter wheel containing filters at wavelengths of 340 and 380 nm. Cells were alternately excited and emission signals of fura 2-loaded cells were collected by a photomultiplier and recorded on-line on a computer screen. As a model system, the rat C-cell carcinoma cell line rMTC 6-23 secreting calcitonin was used. An acute elevation of extracellular calcium resulted in an increase in [Ca2+]i within 5 sec and rapid release of preformed calcitonin.

If you want comparative values, rather than absolute, there is a way of estimating them. Use calcium orange, according to the procedure we showed. Heckman, C. A., Ademuyiwa, O. M., & Cayer, M. L. (2022). How filopodia respond to calcium in the absence of a calcium-binding structural protein: non-channel functions of TRP. Cell communication and signaling : CCS, 20(1), 130. https://doi.org/10.1186/s12964-022-00927-y

To measure the concentration of free calcium in the cytoplasm of rat beta cells, you can use fluorescence-based calcium indicators. Here's a general procedure:

1. Select a Calcium Indicator: Choose a fluorescent calcium indicator that is suitable for your experimental setup. Popular calcium indicators include Fluo-4, Fura-2, and Indo-1. These indicators exhibit changes in fluorescence intensity or ratio upon binding to calcium ions.

2. Loading the Cells: Isolate rat beta cells and culture them in appropriate conditions. Incubate the cells with the calcium indicator by adding the indicator dye to the culture medium. The loading time and concentration of the calcium indicator may vary depending on the specific indicator used and the experimental conditions. Follow the manufacturer's instructions or established protocols for the loading procedure.

3. Cell Imaging: Transfer the loaded cells to a microscope equipped with appropriate filters and imaging capabilities for fluorescence detection. Ensure that the imaging system is capable of excitation at the appropriate wavelength for the calcium indicator you are using.

4. Image Acquisition: Set up the microscope imaging parameters, such as exposure time, image resolution, and acquisition intervals. Acquire images of the cells under basal conditions and during your experimental manipulations (e.g., exposure to various stimuli or drugs). It is advisable to capture images in multiple regions of interest to obtain a representative measurement.

5. Data Analysis: Analyze the acquired images to determine the changes in fluorescence intensity or ratio, which correlate with changes in calcium concentration. Calculate the fluorescence intensity or ratio values for each region of interest using image analysis software. Subtract background fluorescence if necessary.

6. Calibration: Perform a calibration step to convert the fluorescence signal into calcium concentration values. This involves creating a calibration curve by treating the cells with ionomycin (a calcium ionophore) and measuring the fluorescence or ratio response at different known calcium concentrations. Fit the calibration data with an appropriate mathematical model to obtain a conversion equation for calcium concentration estimation.

7. Calcium Concentration Measurement: Apply the calibration equation to the fluorescence or ratio values obtained from the experimental measurements to determine the corresponding calcium concentrations in the cytoplasm of the beta cells.

Thank you very much for your guidance

In fact, I do not intend to isolate the beta cell. I want to check the changes in calcium concentration in the islands. In fact, I want to see when the islets are exposed to glucose concentrations of 6.5 and 17.6 millimolar and we measure the amount of insulin secretion in these two conditions, how will the calcium concentration of the islets change.

I see. It seems that you are interested in studying the changes in calcium concentration in pancreatic islets when exposed to different glucose concentrations and how it relates to insulin secretion. Calcium plays a crucial role in the regulation of insulin release from pancreatic beta cells within the islets of Langerhans.

When glucose levels rise, it triggers a series of events in beta cells that lead to insulin secretion. One of these events is an increase in intracellular calcium concentration. Here's a general overview of the process:

By manipulating the glucose concentrations to 6.5 and 17.6 millimolar, you can observe how these different glucose levels affect the calcium concentration in the islets and subsequently measure the amount of insulin secretion.

Typically, experiments like these involve using fluorescent calcium indicators, such as Fura-2 or Fluo-4, which can selectively bind to calcium ions and emit fluorescence when calcium is present. These indicators can be loaded into the islets, allowing real-time monitoring of changes in calcium concentration.

By exposing the islets to the desired glucose concentrations and using calcium imaging techniques, you can quantify the changes in calcium concentration within the islets and correlate them with insulin secretion measurements. This will help you understand how different glucose levels influence calcium dynamics and subsequently affect insulin release from the pancreatic islets.

It's worth mentioning that studying islets in their intact form offers a more physiologically relevant context compared to isolated beta cells alone, as islets contain multiple cell types that interact and contribute to glucose sensing and insulin secretion.

I hope this clarifies the approach you can take to investigate the changes in calcium concentration in pancreatic islets when exposed to different glucose concentrations. If you have any further questions, feel free to ask!

Thank you very much for your time and consideration