Prolonged Retention of Calbryte™ 520 AM for In Vitro Analysis
Calcium-imaging is a sensitive method for monitoring the spatiotemporal dynamics of calcium signaling. Since intracellular concentrations are correlated with physiological and pathophysiological characteristics of the central nervous system (CNS), calcium imaging with fluorescent indicators provides an ultra-sensitive and accurate method for measuring calcium responses in vitro. In this technical note, we compared the performance of two fluorescent calcium indicators - Fluo-4 AM and Calbryte™ 520 AM - following prolong incubation periods post cell loading and measured the response upon ATP stimulation. Cells loaded with Calbryte™ 520 AM retained the dye for 24 hours post staining while exhibiting superior signal-to-noise characteristics. The longer retention of Calbryte™ 520 AM can be exploited to accomodate for the extended time required when monitoring calcium dynamics in acute neuronal tissue.
Calcium is an important secondary messenger that plays a key role in different intracellular physiological processes, and due to its importance in regulating such processes, calcium imaging is widely used in many laboratories all over the world. The source of calcium entry to the cytoplasm can be either external or internal, and its concentration is tightly maintained by calcium transporters that remove calcium ions rapidly to avoid cytotoxic effects. Cytosolic Ca+2 signals are generally classified as being either transient, sustained or oscillatory depending upon the degree and duration of calcium influx. To satisfy these variations in calcium signaling, AAT Bioquest has pioneered major advancements in calcium detection by developing a series of indicators with wider wavelength options, brighter fluorescence intensities and better photostability enabling faster, more accurate measurements of calcium concentration and mobilization.
One limitation that has proved challenging in calcium imaging is the polar nature of calcium indicators. By their nature, negatively charged compounds are cell-impermeant and require either physically invasive loading techniques, such as microinjection, electroporation, or patch pipette, or chemical loading techniques to enter live cells. The latter, which was first introduced by Roger Tsien, offers a more convenient and passive method for loading hydrophilic dyes into live cells. In order to chemically load dyes, they must be first modified with acetoxymethyl (AM) esters. AM ester modification masks the negatively charged carboxylic groups and produces uncharged hydrophobic indicators, which can be passively loaded into live cells. Following hydrolysis of the AM groups by intracellular proteases, indicators are released and stay inside the cells. However, the loading efficiency of calcium indicators varies between cell types due to organic anion transporters that can extrude the dye. For that reason, probenecid, which is an organic anion transporter inhibitor, is used to improve dye retention by deactivating these pumps. Unfortunately, probenecid is not without caveats. Probenecid has been shown to adversely affect platelet aggregation and cytotoxicity, processes that involve intracellular Ca+2 homeostasis , and calcium flux kinetics of cyclic nucleotide gated ion channels resulting in reducing response of the agonists .
Here, we describe a novel indicator for calcium imaging applications, Calbryte™ 520. This indicator is part of the Calbryte™ series, a family of fluorescent dyes developed to monitor intracellular calcium. Calbryte™ 520 absorbs light at 493 nm and emits at 515 nm, making it spectrally similar to Fluo-4 (Ex/Em = 495/528 nm). However, Calbryte™ 520 is far superior than Fluo-4, particularly when comparing dye-retention and signal-to-noise ratios. In the present study, Calbryte™ 520 AM and Fluo-4 AM were loaded into cells under the same conditions. ATP-induced calcium responses were measured using a fluorescence microscope after 0 hours, 4 hours and 24 hours of prolonged incubation. Comparisons were made for fluorescence intensity, signal-to-noise ratio, and dye retention. Additionally, Calbryte™ 520 was used to measure changes in calcium levels by flow cytometry after 0 hours, 24 hours and 48 hours of incubation.
Materials and Methods
CHO-K1 cells were cultured at 37°C in a F12-K nutrient mixture containing heat inactivated serum (5% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin), under a humidified atmosphere containing 5% CO2. HeLa cells were cultured at 37°C in DMEM containing heat inactivated serum (10% Fetal Bovine Serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin), under a humidified atmosphere containing 5% CO2. Jurkat cells were cultured at 37°C in RPMI containing heat inactivated serum (10% Fetal Bovine Serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin), under a humidified atmosphere containing 5% CO2.
Fluorescence microscopy Calbryte™ 520 AM and Fluo-4 AM
Cells were plated in 100 µL culture medium in a 96-well black wall/clear bottom costar plate (Greiner Bio-One, Kremsmunster, Austria) at 50,000 cells per well. The next day, equal volumes of Fluo-4 AM (AAT Cat# 20550) and Calbryte™ 520 AM (AAT Cat# 20650)-loading solutions with final concentrations of 5 µM and 0.02% PF-127 (AAT Cat# 20053) in the absence of a probenecid were added to each well. Cells were incubated with the dye-loading buffer for 120 minutes at 37°C, 5% CO2 incubator. The dye loading solution was then removed, fresh cell culture medium (200 µL) was added to the plate and incubated at 37°C, 5% CO2 incubator. Response was measured at the indicated times by adding 5X solution of ATP to stimulate P2Y1 signaling. Images were acquired with fluorescence microscopes (Keyence) using the FITC filter set.
Flow cytometry analysis for longer retention tests
Cells were plated in 10-cm flask at 50,000 cells per well. The next day, equal volumes of Calbryte™ 520 AM-loading solutions with final concentrations of 5 µM were added to each well and incubated for 60 minutes at 37°, 5% CO2 incubator. The dye loading solution was then removed, fresh cell culture medium was added to the plate and incubated at 37°, 5% CO2 incubator. The cells were collected at indicated intervals and washed once with HH buffer. Flow cytometry was performed with FITC channel using NovoCyte® flow cytometer from ACEA Biosciences.
Retention test for Calbryte™ 520 AM in Jurkat cells using flow cytometry. Jurkat cells were stained with Calbryte™ 520 AM at 5 µM for 1 hour at 37°C incubator. Cells were washed once and grown in cell culture medium and subjected to flow cytometry analysis at indicated times. The response was recorded using the FITC channel in NovoCyte flow cytometer. Data shown are representative of at least 3 biological replicates.
Prolonged retention of Calbryte™ 520 AM
Flow cytometry analysis was performed to assess Calbryte™ 520 AM retention in cells following prolonged periods of incubation. Jurkat cells were stained with Calbryte™ 520 AM, washed and then grown in cell culture medium for several days. After a period of 2 days, it was observed that Calbryte™ 520 AM was well-retained in cells (Figure 1), making it a suitable choice for long-term calcium tracing in vitro.
Calcium detection after a prolonged retention in cells
This study aimed to demonstrate the length of time cells, loaded with calcium indicators, can be maintained with the dye to produce reliable and reproducible data about cell function. We loaded the HeLa (See supplemental figure 1) and CHO-K1 (Figure 2) cells with Fluo-4 AM and Calbryte™ 520 AM in the absence of probenecid and incubated for 120 minutes at 37°C, 5% CO2 incubator. After incubation, cells were shifted to full cell culture medium and stimulated P2Y signaling using ATP solution at indicated times. We showed that adding ATP solution will stimulate the cells and induce fluorescence in Calbryte™ 520 AM containing cells post loading up to 24 hours but the same response was not observed in Fluo-4 AM containing samples possibly due to its poor retention property.
Left: Response of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were stained with Fluo-4 AM or Calbryte™ 520 AM in HHBS without probenecid, and the cells were incubated at 37°C for 2 hours. After that, cells were grown in the cell culture medium. At indicated times, the cell culture medium was replaced with 200 µL HHBS and 50 µL of 50 µM ATP were added, and imaged with a fluorescence microscope (Keyence) using FITC channel. Right: Response of endogenous P2Y receptor to ATP in HeLa cells. HeLa cells were stained with Fluo-4 AM or Calbryte™ 520 AM in HHBS without probenecid, and the cells were incubated at 37°C for 2 hours. After that, cells were grown in the cell culture medium. At indicated times, the cell culture medium was replaced with 200 µL HHBS and 50 µL of 50 µM ATP were added, and imaged with a fluorescence microscope (Keyence) using FITC channel.
Our comparative study demonstrated the cells loaded with Calbryte™ 520 AM were capable of retaining the indicator for prolonged periods of time (over 48 hours) without the use of probenecid. The longer duration of staining would immensely help staining the tissue samples which requires longer workaround time.
Product Ordering Information
Table 1. Calbryte™ 520 green fluorescent calcium indicators for live cell calcium imaging.
|Calbryte™ 520 AM||493||515||FITC||1.2 µM||0.75||∼300 fold||2x50 µg||20650|
|Calbryte™ 520 AM||493||515||FITC||1.2 µM||0.75||∼300 fold||10x50 µg||20651|
|Calbryte™ 520 AM||493||515||FITC||1.2 µM||0.75||∼300 fold||1 mg||20653|
|Calbryte™ 520, potassium salt||493||515||FITC||1.2 µM||0.75||∼300 fold||10x50 µg||20658|
|Calbryte™ 520, potassium salt||493||515||FITC||1.2 µM||0.75||∼300 fold||2x50 µg||20656|
Table 2. Probenecid and Pluronic® F-127 products for improving calcium indicator retention and aqueous solubility.
|ReadiUse™ probenecid *25 mM stabilized aqueous solution*||10x10 mL||20062|
|ReadiUse™ probenecid, sodium salt *Water-soluble*||10x77 mg||20061|
|Probenecid *Cell culture tested* *CAS 57-66-9*||10x72 mg||20060|
|Pluronic® F-127 *20% solution in DMSO*||10 mL||20052|
|Pluronic® F-127 *Cell culture tested *||10 g||20050|
|Pluronic® F-127 *10% solution in water*||10 mL||20053|
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