# **Real-Time Monitoring of Intracellular Ca2+ Dynamics in HEK293 Cells with Celloger® Pro** ## **Introduction** Intracellular calcium (**Ca2+**) signaling plays a pivotal role in regulating diverse cellular processes, including **neurotransmission**, **secretion**, **proliferation**, and **apoptosis**. Monitoring these rapid and transient calcium dynamics in real time is essential for understanding signal transduction pathways and cellular responses to external stimuli. To visualize such calcium fluxes, **genetically encoded calcium indicators (GECIs)** have emerged as powerful alternatives to traditional chemical dyes. Among them, **GCaMP3**—a fusion protein composed of circularly permuted green fluorescent protein (**cpGFP**), **calmodulin (CaM)**, and the **M13 peptide**—undergoes a calcium-dependent conformational change that enhances fluorescence intensity. Compared to synthetic dyes, **GCaMP3** offers improved sensitivity and compatibility, lower cytotoxicity, and suitability for long-term live-cell imaging. In this application note, **HEK293 cells** were selected as the model system because of their ease of culture, high transfection efficiency, and robust calcium signaling capacity. Although not excitable like neurons, HEK293 cells respond readily to chemical stimulation with measurable calcium transients, providing a practical platform for validating calcium imaging workflows. To induce calcium signaling, two well-characterized agonists were employed: **ATP (purinergic receptor agonist)** and **histamine (H1 receptor agonist)**. By applying these stimuli to GCaMP3-expressing HEK293 cells, we demonstrate how **Celloger® Pro** enables real-time monitoring and analysis of distinct intracellular **Ca2+** dynamics. --- ## **Materials and Methods** ### **GCaMP3 Transfection** 1. **Cell Seeding:** HEK293 cells were seeded in a 12-well plate at **1.5×10⁵ cells/well** in **Dulbecco’s Modified Eagle Medium (DMEM)** supplemented with **10% fetal bovine serum (FBS)** and **1% penicillin-streptomycin**. 2. **Transfection:** **GCaMP3 plasmid** transfection was performed using **Lipofectamine™ 3000 (Thermo Fisher Scientific, L3000001)** in **Opti-MEM™ (Thermo Fisher Scientific, 31985062\)** according to the manufacturer’s instructions. 3. After **3 hours**, the medium was replaced with fresh complete DMEM, and cells were incubated for an additional **24 hours**. 4. To enrich GCaMP3-expressing cells, **G-418 (Merck, 108321-42-2, 500 µg/mL)** selection was applied for **6 days** with medium changes every **2–3 days**. 5. Following selection, cells were transferred to a 24-well plate and incubated overnight. ### **ATP/Histamine Stimulation and Imaging** 1. **Imaging Conditions:** Intracellular Ca2+ imaging with **Celloger® Pro** was performed at room temperature. 2. **Preparation:** Culture medium was removed, and cells were washed once with **PBS** to eliminate residual calcium. 3. After mounting the plate on the device and adjusting the focus in the green fluorescence channel, either **ATP (100 µM)** or **histamine (100 µM)** was added manually as Preview Recording began. 4. Each recording lasted approximately **1 minute** and was analyzed using **ImageJ**. 5. **ROI-based measurements** were performed frame by frame to quantify changes in green fluorescence intensity (**∆F/F₀**). --- ## **Results** The schematic in **Figure 1A** illustrates the overall experimental workflow and the GCaMP3 activation mechanism. When chemical agonists bind to their cognate receptors, intracellular Ca2+ rises either by influx across the plasma membrane or by release from the endoplasmic reticulum (**ER**). Upon Ca2+ binding, **GCaMP3 fluorescence increases**, enabling real-time observation and recording of intracellular Ca2+ dynamics. Representative fluorescence images (**Figure 1B**) show Ca2+-dependent fluorescence changes in HEK293 cells following ATP treatment. Notably, **ATP stimulation** induced a sharp rise in fluorescence intensity at around **7 seconds**, followed by a gradual decline by **30 seconds**. In contrast, untreated cells exhibited a slow decrease in fluorescence over the same period, presumably due to photobleaching from continuous light exposure. To further characterize stimulus-specific signaling patterns, intracellular Ca2+ responses were quantitatively compared under four conditions: * **Control (CTR)** * **ATP** * **Histamine** * **ATP in Ca2+-free DPBS \[ATP (-Ca2+)\]** Histamine was included as a comparative agonist, whereas the ATP (-Ca2+) condition assessed responses in the absence of extracellular Ca2+. Normalized GCaMP3 fluorescence intensity was calculated using the following equation: ∆F/F0=(Ft−F0)/F0∆F/F₀ \= (Fₜ \- F₀) / F₀∆F/F0​=(Ft​−F0​)/F0​ Where: * **Fₜ** \= fluorescence intensity at time t * **F₀** \= baseline fluorescence intensity at 0 s As shown in **Figure 2A**, **ATP** triggered a rapid and robust increase in GCaMP3 fluorescence, whereas **histamine** produced a slower and less intense signal. The control showed negligible changes over time. These differences were further supported by peak fluorescence intensity measurements (**Figure 2B**). The observed patterns reflect the underlying signaling mechanisms: * **ATP** activates purinergic **P2 receptors**: P2X channels drive immediate Ca2+ influx, while P2Y receptors initiate **IP3-dependent Ca2+ release**, leading to rapid and robust signaling. * **Histamine** primarily signals through **Gq-coupled H1 receptors** in HEK293 cells, eliciting slower **IP3/Ca2+ signaling**. Interestingly, **ATP in Ca2+-free DPBS** exhibited distinct kinetics, showing delayed and modest increases in fluorescence intensity (**Figure 2A–C**). Compared to ATP under normal Ca2+ conditions, the response was minimal due to the lack of extracellular Ca2+ influx. However, fluorescence intensity remained slightly higher than in the control, suggesting ATP may have triggered Ca2+ release via **IP3-mediated ER signaling**. Together, these results demonstrate that **ATP and histamine** activate distinct receptor pathways with different Ca2+ signaling profiles in HEK293 cells. The use of Ca2+-free buffer further highlights the contribution of intracellular Ca2+ stores. --- \[Image placeholder: **Figure 1\. Real-time Ca2+ imaging of GCaMP3-expressing HEK293 cells during ATP stimulation** (A) Schematic of the experimental workflow. HEK293 cells were transfected with GCaMP3, a genetically encoded calcium indicator whose fluorescence increases upon Ca2+ binding. After incubation, cells were treated with ATP or histamine and monitored in real time with the **Celloger® Pro** system. The Preview Recording mode was initiated concurrently with agonist addition to capture rapid changes in intracellular Ca2+ levels. (B) Representative green fluorescence images showing responses to **ATP (100 µM)** at 0, 7, and 30 seconds after addition. White arrows indicate cells with no detectable change in fluorescence; yellow arrows indicate cells with distinct changes in fluorescence intensity. **Scale bars:** 200 µm.\] --- \[Image placeholder: **Figure 2\. Quantitative analysis of intracellular Ca2+ responses** Intracellular Ca2+ responses were quantitatively analyzed under four conditions: unstimulated control (CTR), ATP, histamine, and ATP in Ca2+-free DPBS \[ATP (-Ca2+)\]. (A) Time-course plots show normalized fluorescence intensity (∆F/F₀) over the recording period. (B) Peak fluorescence intensity is presented for each condition (arbitrary units, a.u.). (C) Latency to maximum response is compared among ATP, histamine, and ATP(-Ca2+), while the control is excluded because no distinct peak is observed. Statistical significance was evaluated using **one-way ANOVA** followed by **Tukey’s multiple-comparisons test** (\*p\<0.05, \*\*p\<0.01, \*\*\*p\<0.0001).\] --- ## **Conclusion** In this study, **GCaMP3-based Ca2+ imaging** in HEK293 cells was employed to investigate intracellular calcium signaling in response to chemical stimulation. The temporal patterns of cellular responses varied depending on the stimulant, with distinct differences in kinetics and peak intensities. Notably, **ATP induced a measurable fluorescence increase even under Ca2+-free conditions**, suggesting mobilization from intracellular stores. Using **Celloger® Pro**, these rapid and transient Ca2+ responses were effectively monitored in real time. The system provides a seamless workflow for live-cell imaging and preview recording, enabling researchers to characterize **stimulus-specific signaling dynamics** with precision. Collectively, these findings underscore the importance of **time-resolved Ca2+ imaging** for accurate interpretation of intracellular signaling events at the single-cell level. --- ## **References** 1. Bootman, D. M. (2020). *Fundamentals of Cellular Calcium Signaling: A Primer.* Cold Spring Harbor Perspectives in Biology, 12(1). 2. Oh, J. 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