Calcein(AR)
(Synonyms: Fluorescein Complexone, NSC 298193) 目录号 : GC62882
Calcein 是一种荧光染料和自灭探针，可以作为脂囊泡渗漏的指示剂，也可作 EDTA 钙离子滴定和荧光测定钙离子的络合指示剂。
Cas No.:1461-15-0
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Coelomocytes suspensions (ISO and CF) are separately incubated with calcein-AM (excitation and emission wavelength: 496 nm and 516 nm, respectively) at a final concentration of 200 nM, during 30 min, at 26°C. Calcein-AM is a nonfluorescent ABC transporter substrate whose intracellular accumulation is inversely proportional to ABC transporter activity. Intracellular esterases convert calcein-AM into the fluorescent dye calcein, which is not an ABC transporter substrate, thereby accumulating the dye inside the cell. Therefore, a high fluorescence signal indicates low ABC transporter activity whereas a low fluorescence signal indicates high activity. The fluorescence of samples is measured by flow cytometer. The experiment is repeated six times in duplicates.
[1]. Marques-Santos LF, et al. ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas. Fish Shellfish Immunol. 2017 Sep 5;70:195-203.
Calcein is a fluorescent dye and self-quenching probe, used as an indicator of lipid vesicle leakage, and also as a complexometric indicator for titration of calcium ions with EDTA, and for fluorometric determination of calcium.
Calcein accumulates intracellularly in coelomocytes incubated in coelomic fluid or ISO-EDTA. Coelomocytes incubated in CF show an increase in the calcein fluorescence intensity compared to the control.
[1]. Marques-Santos LF, et al. ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas. Fish Shellfish Immunol. 2017 Sep 5;70:195-203.
| Cas No. | 1461-15-0 | SDF | |
| 别名 | Fluorescein Complexone, NSC 298193 | ||
| 分子式 | C30H26N2O13 | 分子量 | 622.53 |
| 溶解度 | DMSO : ≥ 100 mg/mL (160.63 mM)|0.1 M NaOH : 10 mg/mL (16.06 mM; ultrasonic and adjust pH to 11 with NaOH) | 储存条件 | 4°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.6063 mL | 8.0317 mL | 16.0635 mL |
| 5 mM | 0.3213 mL | 1.6063 mL | 3.2127 mL |
| 10 mM | 0.1606 mL | 0.8032 mL | 1.6063 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters
Macromol Biosci 2022 Oct;22(10):e2200167.PMID:35933579DOI:10.1002/mabi.202200167.
Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are established analyzing the performance of these nanocarriers. Among them, the release of the membrane-impermeable dye Calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the Calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges, and limitations of the assay, and a brief summary of complementary methods. Based on this review, it is hoped to encourage further research groups to take advantage of the Calcein release assay for their own purposes and help to create a database for more efficient cross-correlations between nanocarriers.
Calcein Release from Cells In Vitro via Reversible and Irreversible Electroporation
J Membr Biol 2018 Feb;251(1):119-130.PMID:29143077DOI:10.1007/s00232-017-0005-8.
The aim of this study was to investigate the dependence of Calcein extraction and cell viability on the parameters of pulsed electric field (PEF). Two different approaches concerning PEF parameters were investigated: (1) extraction efficiency and cell viability dependence on pulse number, exploiting 1200 V/cm 100 µs duration high voltage (HV) electric pulses and (2) extraction efficiency and cell viability dependence on the pulses with different duration (44-400 µs) and electric field strength (600-1800 V/cm) that result in the same amount of electric field energy delivered to Chinese hamster ovary cells. Extraction efficiency was evaluated as a percentage ratio of Calcein fluorescence intensity prior and after PEF treatment. Cell viability was evaluated using PI test and cell clonogenic assay. Moreover, Calcein release dynamics from cells after 600 V/cm 400 µs, 1200 V/cm 100 µs, and 1800 V/cm 44 µs was evaluated. Our results show that HV pulses induce instant Calcein extraction due to reversible electroporation; however, subsequent Calcein leakage over time was only observed when 9 HV pulses of 1800 V/cm 44 µs were used. The increased number of pulses resulted in more efficient total Calcein extraction. With the same total energy delivered via electric pulses, the increase of Calcein extraction efficiency was more dependent on pulse strength rather than pulse duration. The highest Calcein extraction efficiency (84.5 ± 7.4%) was obtained using 9 electric field pulses of 1800 V/cm, 44 µs at 1 Hz. Furthermore, the extraction efficiency can be significantly enhanced if external mechanical stress (pipetting) is applied to cells. Cell viability was determined to be dependent on different PEF exposure parameters. It varied from 96.8 ± 4.8 to 31.2 ± 8.9%, implying the possibility to adjust PEF parameter combinations to maintain high cell viability.
Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells
STAR Protoc 2020 Nov 2;1(3):100157.PMID:33377051DOI:10.1016/j.xpro.2020.100157.
Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).
Calcein+/PI- as an early apoptotic feature in Leishmania
PLoS One 2017 Nov 7;12(11):e0187756.PMID:29112976DOI:10.1371/journal.pone.0187756.
Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of Calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.
Light Activation of Calcein Inhibits Vesicle Release of Catecholamines
ACS Chem Neurosci 2017 Oct 18;8(10):2309-2314.PMID:28707873DOI:10.1021/acschemneuro.7b00225.
Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that Calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when Calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of Calcein. We hypothesized that Calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when Calcein is protected from light during experimentation, little to no reduction of vesicle secretion occurred. Therefore, photoactivated Calcein can cause deleterious results for measurements of cellular processes, likely to be the result of release of reactive oxygen species.