Calcein-AM (Calcein acetoxymethyl ester)
(Synonyms: 钙黄绿素-AM; Calcein acetoxymethyl ester) 目录号 : GC34061
Calcein-AM是一种高脂溶性的活细胞染料，能够快速进入存活的细胞。
Cas No.:148504-34-1
Sample solution is provided at 25 µL, 10mM.
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Cell viability was tested by Calcein-AM/PI Double Staining Kit. 2 mL of PBS solution containing 0.1 vol% Calcein AM(GlpBio) and 0.1 vol% PI was added into the samples and incubated for 30 min at 37 °C.
Small Methods (2024): 2400223. PMID: 38602202 IF: 12.4002 -
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MSC coculture inhibits neuronal ferroptosis through mitochondrial transfer.(K) Flow cytometry analysis of LIP in Ht22 cells from culture alone, coculture with MSCs or MSC coculture combined with Cyto D treatment after Rsl3 stimulation for 24 h. Cells were incubated by Calcein-AM (FITC channel) and treated with or without Deferiprone.
After trypsinization, cells (6-well dishes) were washed twice with PBS and then incubated with 1 μM Calcein-AM(GlpBio) at 37°C for 15 min.
Redox Biology, 2023: 102871. PMID: 37699320 IF: 11.3997 -
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PEF inhibits ferroptosis in C. elegans models of AD.(L) Representative images of CL4176 worms stained with Calcein-AM reagent in the presence or absence of PEF (800 μg/mL) and DFO (100 μM). (M) The bar chart shows the relative Calcein-AM intensity in CL4176 worms.
Briefly, after treatment, worms were collected and co-cultured in M9 with 0.05 μg/mL Calcein-AM(GlpBio) for 1 hour, followed by three washes in 1 mL M9.
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Related Biological Data
NAR reduced cellular iron concentration. (A) Chondrocytes were treated with IL-1β and FAC, with or without various concentrations of NAR for 24 hr, and then stained with a Calcein-AM fluorescent probe for 15min.
Chondrocytes were treated with IL-1β and FAC, with or without various concentrations of NAR for 24 hr, and then stained with a Calcein-AM(GlpBio) fluorescent probe for 15min.
Phytomedicine (2022): 154330. PMID: 35905566 IF: 6.6563 -
Related Biological Data
Tagitinin C induces ferroptosis in HCT116 cells.(G) Quantification of cellular LIP levels using the calcein-AM (C-AM) method. The mean fluorescence intensity (MFI) of C-AM is subtracted from the MFI of C-AM treated with DFO.
After trypsinization, the cells were washed twice with PBS followed by incubation of 2 µM calcein-acetoxymethyI ester (GLPBIO) for 30 min at 37 °C.
Int J Biol Sci 17.11 (2021): 2703-2717. PMID: 34345202 IF: 6.58
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| Cell experiment [1]: | |
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Cell lines |
EPC cells |
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Preparation Method |
Uptake kinetics of EPC cells loaded with 5 µM calcein AM seeded at 1x105 cells well-1 and cultured at 15 °C. Calcein AM uptake was measured as fluorescence intensity (FI). |
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Reaction Conditions |
5 µM at 15 °C, 1-8h |
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Applications |
Uptake kinetics showed that for EPC cells seeded at a density of 1x105 cells well-1 calcein AM labelling increased throughout the 7 h tested. |
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References: [1]. Iwanowicz LR, et al. Calcein AM release-based cytotoxic cell assay for fish leucocytes. Fish Shellfish Immunol. 2004 Feb;16(2):127-37. |
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Calcein-AM is a highly lipophilic vital dye that rapidly enters viable cells. It is converted by intracellular esterases to calcein that produces an intense green (530-nm) signal, and is retained by cells with intact plasma membrane.[1]
In vitro experiment it shown that calcein-AM assay used to assess human RBC viability after incubation (37°C for 3 and 20 h) in the presence of Ca2+ (2.5 mM) and ionophore A 23187 (0.5 μM).[1] 0.05 μM was the optimal concentration of CAM (Calcein-AM) for staining effector cells by testing 0.05, 0.1, 0.2, and 0.4 μM. Using 0.05 μM CAM to stain the PBMCs and expanded NK cells from three normal volunteers, the results demonstrated that there is no significant decrease in cytotoxicity and CAM staining had no significant effect on human NK cell activity in PBMCs or in expanded NK cells.[2] In vitro, 50μm calcein AM's fluorescent signal of 1 x lo5 lymphocytes was close to the saturation level, while the signal emitted by lymphocytes labeled with 20μm calcein AM was only slightly lower. [3] Calcein-AM has cytotoxic activity against human tumor cell lines (such as the human lymphoma U-937-GTB) at low concentrations (2.5 ug/ml).[4] In vitro experiment it demonstrated that in the mixed macrophages and THP-1 cells (5x105 cells/ml), Calcein-AM (2 µM)/propidium Iodide (PI) (4.5 µM) staining assay Calcein-AM/PI double staining was used to quantify the number of living and dead cells as a cell death assay.[5] In addition, The cells in OA chondrocytes were seeded in 24-well plates (2 × 104 cells/well), cultured for 4 h, the cells were treated with 5 μL Calcein-AM (2 μM) and 5 μL PI (2 μM) at 37°C in conditions void of light for 30 min, and then analyzed under a fluorescence microscope.[6]
References:
[1].Bratosin D, et al. Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. Cytometry A. 2005 Jul;66(1):78-84.
[2].Jang YY, et al. An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells. Ann Clin Lab Sci. 2012 Winter;42(1):42-9.
[3].Braut-Boucher F, et al. A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes. J Immunol Methods. 1995 Jan 13;178(1):41-51.
[4].Liminga G, et al. Cytotoxic effect of calcein acetoxymethyl ester on human tumor cell lines: drug delivery by intracellular trapping. Anticancer Drugs. 1995 Aug;6(4):578-85.
[5].Xiang N, et al. Gardnerella vaginalis induces NLRP3 inflammasome-mediated pyroptosis in macrophages and THP-1 monocytes. Exp Ther Med. 2021 Oct;22(4):1174.
[6].Zhang L, et al. MicroRNA-140-5p represses chondrocyte pyroptosis and relieves cartilage injury in osteoarthritis by inhibiting cathepsin B/Nod-like receptor protein 3. Bioengineered. 2021 Dec;12(2):9949-9964.
Calcein-AM是一种高脂溶性的活细胞染料,能够快速进入存活的细胞。它被细胞内酯酶转化为卡尔西因,产生强烈的绿色（530纳米）信号,并被具有完整质膜的细胞保留。
实验室内的实验证明,使用荧光染料Calcein-AM可以评估人类红细胞在存在Ca2+（2.5 mM）和离子载体A 23187（0.5 μM）下孵育后（37°C,3小时和20小时）的存活率。测试了0.05、0.1、0.2和0.4μM浓度的CAM,结果显示0.05μM是染色效果最佳的浓度。使用0.05μM CAM对三名正常志愿者PBMCs和扩增NK细胞进行染色,结果表明,在PBMCs或扩增NK细胞中,Cytotoxicity没有显著降低,并且CAM染色对人类NK细胞活性没有显著影响。在体外实验中,50μm Calcein AM荧光信号接近饱和水平时1 x lo5淋巴细胞标记物发出的信号略微较低。Calcein-AM对人类肿瘤细胞系（如人淋巴瘤U-937-GTB）具有毒性作用,在低浓度下（2.5ug/ml）。在混合巨噬细胞和THP-1 细胞 (5x105 cells/ml) 的体外实验中, 使用 Calcein-AM(2 µM)/propidium Iodide (PI) (4.5 µM) 染色法进行 Calcein-AM/PI 双重染色,以定量活细胞和死亡细胞的数量作为细胞死亡检测方法。此外,在OA软骨细胞中种植2×104个细胞/well,培养4小时后,在无光条件下将5μL Calcein-AM（2μM）和5μL PI（2μM）处理在37°C下30分钟,并在荧光显微镜下分析。
| Cas No. | 148504-34-1 | SDF | |
| 别名 | 钙黄绿素-AM; Calcein acetoxymethyl ester | ||
| 化学名 | tetrakis(acetoxymethyl) 2,2',2'',2'''-(((3',6'-diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-4',5'-diyl)bis(methylene))bis(azanetriyl))tetraacetate | ||
| Canonical SMILES | O=C1OC2(C(C=C(CN(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)C(OC(C)=O)=C3)=C3OC4=CC(OC(C)=O)=C(CN(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)C=C24)C5=C1C=CC=C5 | ||
| 分子式 | C46H46N2O23 | 分子量 | 994.86 |
| 溶解度 | 10 mg/mL in EtOH, MeOH, DMSO, DMF with gentle heating (37°C) for 10 minutes. | 储存条件 | -20°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 | 1.0052 mL | 5.0258 mL | 10.0517 mL |
| 5 mM | 0.201 mL | 1.0052 mL | 2.0103 mL |
| 10 mM | 0.1005 mL | 0.5026 mL | 1.0052 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。
Cytotoxic activity of Calcein acetoxymethyl ester (Calcein/AM) on primary cultures of human haematological and solid tumours
Eur J Cancer 1996 May;32A(5):883-7.PMID:9081371DOI:10.1016/0959-8049(96)00015-9.
The aim of this study was to determine the in vitro cytotoxicity of Calcein acetoxymethyl ester (Calcein/AM) on primary cultures derived from solid and haematological human tumours. Calcein/AM is a fluorescent dye that localises intracellularly after esterase-dependent cellular trapping and which has shown cytotoxic activity against various established human tumour cell lines at relatively low concentrations. The semi-automated fluorometric microculture cytotoxicity assay, based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate to fluorescein, in microtitre plates was used for the evaluation of Calcein/AM activity in tumour cell suspensions from patients. The cytotoxicity was measured as a survival index (SI), defined as the fluorescence as a percentage of control cultures. A total of 163 evaluable samples from various tumours were tested with continuous drug exposure. The activity of Calcein/AM was compared with representatives of six major classes of standard chemotherapeutic drugs. Calcein/AM was found to induce concentration-dependent decreases in the SI of both haematological and solid tumour cells. The ratio of solid over haematological tumour activity increased at a rate that was concentration dependent. Although it was relatively less active than cisplatin against solid tumours, Calcein/AM showed higher solid tumour activity compared to leukaemic specific agents (cytarabine and amsacrine), vincristine and doxorubicin (Dox). Among the solid tumours tested, childhood tumours, non-small cell lung cancer and sarcomas were the most sensitive to Calcein/AM. The best correlation between SI values was seen between Calcein/AM and Dox, with weaker correlations to representatives of antimetabolites, platinum compounds, topoisomerase II inhibitors, tubulin interactive agents and alkylators. Non-cytotoxic concentrations of cyclosporin A significantly potentiated calcein-induced cytotoxicity. The results show that Calcein/AM is differentially active against haematological tumours, but with substantial activity against solid tumours. The drug may represent a new class of anticancer compound with a unique means of drug delivery.
Cytotoxic effect of Calcein acetoxymethyl ester on human tumor cell lines: drug delivery by intracellular trapping
Anticancer Drugs 1995 Aug;6(4):578-85.PMID:7579562DOI:10.1097/00001813-199508000-00011.
Calcein acetoxymethyl ester (calcein/AM) and some related cellular dyes with a cytoplasmic distribution were investigated with respect to cellular hydrolysis, accumulation, efflux and cytotoxicity in a panel of established human cell lines, including multidrug resistant (MDR) phenotypes. At 0.1-1 micrograms/ml, calcein/AM was highly cytotoxic against several cell lines, even after short-term exposure (30 min). Calcein/AM induced no immediate loss (3 h) of membrane integrity and the drug was more active against low compared with high density plated cells. In cell lines with the MDR phenotype and in the renal carcinoma cell line ACHN, the drug was considerably less active. Non-esterified calcein had no effect and calcein/AM was significantly more potent than other structurally related fluorescein analogs and AM esters tested. Although MDR cell lines showed a decreased cellular hydrolysis and accumulation of the dye, there was no strict relationship between cytoplasmic calcein exposure and cytotoxic activity. The rate of efflux was low in the two most sensitive cell lines, the human lymphoma U-937-GTB and its vincristine (vcr) resistant subline U-937/vcr10, while the remaining cell lines showed similar biphasic efflux patterns, including cell lines of the MDR phenotype. The results show that calcein/AM has cytotoxic activity against human tumor cell lines at low concentrations. The effect appears dependent on the intracellular trapping of the drug, although the specific cellular target remains unknown. Due to its cytotoxic efficacy and unique principle of cellular drug delivery, further investigation of calcein/AM and related compounds as potentially new anticancer agents seems warranted.
Microfluorometric evaluation of Calcein acetoxymethyl ester as a probe for P-glycoprotein-mediated resistance: effects of cyclosporin A and its nonimmunosuppressive analogue SDZ PSC 833
Exp Cell Res 1994 Jun;212(2):291-6.PMID:7910563DOI:10.1006/excr.1994.1146.
A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent Calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.
Cytological Observations of the Large Symbiotic Foraminifer Amphisorus kudakajimensis Using Calcein acetoxymethyl ester
PLoS One 2016 Nov 3;11(11):e0165844.PMID:27812157DOI:10.1371/journal.pone.0165844.
Large benthic foraminifera are unicellular calcifying reef organisms that can form symbiotic relationships with a range of different microalgae. However, the cellular functions, such as symbiosis and calcification, and other aspects of cellular physiology in large benthic foraminifera are not fully understood. Amphisorus kudakajimensis was used as a model to determine the detailed cellular characteristics of large benthic foraminifera. We used Calcein acetoxymethyl ester (calcein AM) as a fluorescent indicator for live confocal imaging. We demonstrated that calcein AM is a useful fluorescent indicator to stain the fine network of reticulopodia and the cytoplasm in living A. kudakajimensis. We showed that at least two types of reticulopodia exist in A. kudakajimensis: the straight bundle of reticulopodia that spreads from the aperture and the fine reticulopodia along the surface of the aperture and chamber walls. The cytoplasm in outer chambers was highly branched and contained a few dinoflagellates. In contrast, the inner chamberlets contained condensed cytoplasm and many dinoflagellates, suggesting that the cytoplasm of A. kudakajimensis performs different functions based on its location within the large test. Our confocal detailed image analysis provides real-time cellular morphology and cell physiology of living foraminifera.
Calcein-acetoxymethy ester enhances the antitumor effects of doxorubicin in nonsmall cell lung cancer by regulating the TopBP1/p53RR pathway
Anticancer Drugs 2017 Sep;28(8):861-868.PMID:28628491DOI:10.1097/CAD.0000000000000527.
Calcein acetoxymethyl ester (Calcein-AM) treatment has been reported to exert antitumor effects in certain cancer cells; however, the detailed mechanism of action of Calcein-AM in cancers remains unclear, especially in nonsmall cell lung cancer (NSCLC). This study focused on the function and mechanism of action of Calcein-AM in NSCLC. We used cell viability assays, western blotting, and EdU proliferation assay combined with Calcein-AM treatment or siRNA interference to investigate the role of topoisomerase IIβ binding protein 1 (TopBP1) and p53 in NSCLC chemotherapy. We found that Calcein-AM has antitumor effects in lung cancer and enhances the antitumor effects of doxorubicin in NSCLC. Furthermore, we found that TopBP1, which we previously showed was involved in doxorubicin resistance through upregulation of aberrant p53, was involved in calcein-AM-mediated increased doxorubicin sensitivity. Doxorubicin upregulated the expression of aberrant p53. Calcein-AM repressed the expression of TopBP1, which resulted in reduced expression of aberrant p53 and disrupted the antiapoptotic activity mediated by the TopBP1/mutp53 pathway in NSCLC. Together, our findings show that Calcein-AM, the cell-permeable derivative of calcein, exerts significant antitumor effects in NSCLC, and can enhance the antitumor effect of doxorubicin by regulating the TopBP1/mutp53 pathway. These findings provide novel insight into lung cancer treatment.