Fluorescent Dye; Emission (Em) = 515; Excitation (Ex) = 489
Substrate for ABC transporters, specifically used as a probe to measure the activity of ABCB1 (P-glycoprotein) and ABCC1 (Multidrug Resistance Protein) like transporters. [1]

In coelomic fluid or ISO-EDTA-incubated coelomocytes, calcein builds up. Calcine fluorescence intensity was higher in coelomic cells cultured in CF as compared to control cultures.

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Calcein-AM is a non-fluorescent molecule that passively diffuses across the cell membrane. Once inside the cell, it is cleaved by intracellular esterases to produce the fluorescent dye calcein. Calcein is not a substrate for ABC transporters and thus accumulates intracellularly. The intracellular accumulation of calcein fluorescence is therefore inversely proportional to the activity of ABC transporters: high fluorescence indicates low transporter activity (efflux), and low fluorescence indicates high transporter activity. [1]
The activity of ABC transporters in sea urchin coelomocytes and oyster hemocytes was assessed by measuring the intracellular accumulation of calcein fluorescence using flow cytometry and fluorescence microscopy. [1]
In experiments with sea urchin (Echinometra lucunter) coelomocytes, a differential pattern of calcein accumulation was observed among cell subpopulations: phagocytes showed the highest fluorescence, followed by colorless spherulocytes, then vibrate cells, and red spherulocytes showed the lowest or absent fluorescence. [1]
Incubation of coelomocytes in natural coelomic fluid resulted in significantly lower calcein accumulation (44.4 ± 3.36 a.u.) compared to incubation in an artificial anticoagulant solution ISO-EDTA (187.1 ± 6.39 a.u.), indicating higher ABC transporter activity in the natural medium. [1]
Treatment with the ABCC1 transporter blocker MK571 (at 10 μM) significantly increased calcein fluorescence in coelomocytes from both sea urchin species (E. lucunter and Echinus esculentus) and in all hemocyte types from both oyster species (Crassostrea gasar and Crassostrea gigas), indicating functional ABCC1-like transporter activity. The increase in fluorescence ranged from 55.7% to 249.2% depending on the cell type and species. [1]
Treatment with the ABCB1 transporter blocker reversin 205 (at 10 μM) significantly increased calcein fluorescence in coelomocytes of E. lucunter and in hyalinocytes of the oyster C. gasar, but had no effect on E. esculentus coelomocytes or other hemocyte types, suggesting species and cell-type specific activity of ABCB1-like transporters. [1]

Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers[1].

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Calcein-AM itself is not an enzyme. It is used as a substrate to measure the functional activity of ABC transporter enzymes (efflux pumps). The assay principle relies on the conversion of Calcein-AM to fluorescent calcein by intracellular esterases, and the subsequent efflux of the non-fluorescent Calcein-AM (or its fluorescent product? The text specifies that calcein is not a substrate, so it's likely the Calcein-AM that is effluxed) by the transporters. Blocking the transporters leads to increased accumulation of the fluorescent calcein. [1]

Examples:
Calcein is a fluorescent probe with yellow green fluorescence, and may be used to determine labile iron pool and measure the Fe(III)-binding affinity with ligands.
Method: For cell staining.
1. Cells (10~6/mL) are loaded by incubation with 20 µM Fe(II) for 2 h at 37°C.
2. Cells are collected by centrifugation (1000 rpm; 5 min) and incubated with Calcein (0.5 µM; 15 min; 37°C) in a D’hanks buffer (pH 7.4).
3. Wash cells for twice by D’hanks buffer (pH 7.4).
4. The fluorescence is monitored by both flow cytometry and a confocal laser scanning microscope.

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Description: Calcein is a fluorescent probe with yellow green fluorescence, and may be used in capillary-like tube formation assay.
Method: For cell staining.
1. Cells are seeded at a density of 5×104 cells/well into Matrigel coated 96-well plates and treated with conditioned medium for 5.5 h under normoxia or hypoxia.
2. Stain cells with Calcein (0.5 h).
3. Tubes forming intact networks are captured by Lion Heart (Biotec) and quantified by counting the number of tubes.

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Description: Calcein is a fluorescent probe with yellow green fluorescence, and may be used in tube formation assay.
Method: For cell staining.
1. Suspend cultured cells in conditioned medium and seeded into Matrigel coated 96-well plates at a density of 5×104 cells/well for 6 h under normoxia or hypoxia.
2. Stain cells with Calcein (10 min).
3. Tubes forming intact networks are captured by Lion Heart and quantified by the tube length and number of branch points using Image J.

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Protocol for sea urchin coelomocytes: Coelomocytes were suspended in either coelomic fluid or ISO-EDTA at a concentration of 1 × 10⁶ cells/mL. The cell suspension was incubated with 200 nM Calcein-AM for 30 minutes at 26°C. For inhibition studies, cells were pre-treated with 10 μM of reversin 205 (ABCB1 blocker) or MK571 (ABCC1 blocker) for 30 minutes prior to the addition of Calcein-AM. After incubation, calcein fluorescence was measured by flow cytometry (FL1 detector, 530/30 nm) or visualized by fluorescence microscopy (excitation filter 510-550 nm). For microscopy, images were captured with a fixed exposure time (500 ms) and fluorescence intensity was quantified using image analysis software. [1]
Protocol for oyster hemocytes: Hemolymph was collected and filtered, and cell concentration was not adjusted. The hemocyte suspension was pre-treated with 10 μM reversin 205 or MK571 for 30 minutes, followed by incubation with 200 nM Calcein-AM for 30 minutes at either 26°C (for C. gasar) or 18°C (for C. gigas). Calcein fluorescence was measured by flow cytometry, and data was analyzed separately for different hemocyte subpopulations (hyalinocytes, granulocytes, blast-like cells) based on their forward and side scatter characteristics. [1]

[1]. 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 fluorescein dye with fluorescent properties, its function being related to fluorescein compounds. Calcein, also known as Fluorexon, is used in ophthalmic solutions as a staining agent for soft or rigid contact lenses. It is a fluorescent dye or luminescent agent.

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Calcein-AM is a non-fluorescent, cell-permeant derivative of calcein. The "AM" (acetoxymethyl) ester groups make the molecule lipophilic, allowing it to cross cell membranes. [1]
Once inside the cell, ubiquitous intracellular esterases cleave the AM groups, producing the hydrophilic, negatively charged, and highly fluorescent calcein, which is retained in the cytosol. [1]
The efflux of Calcein-AM (or potentially the cleaved calcein? The text is clear that calcein is not a substrate, so the efflux is likely of the non-fluorescent Calcein-AM) by ABC transporters competes with its intracellular hydrolysis, making the rate of fluorescence increase a direct measure of transporter activity. [1]
In this study, Calcein-AM was used as a functional probe to demonstrate the presence and activity of ABCB1 and ABCC1-like efflux transporters in the immune cells (coelomocytes and hemocytes) of marine invertebrates from different climatic regions. [1]
The study highlights the importance of using a natural medium (coelomic fluid for sea urchins, hemolymph for oysters) for such assays, as artificial media can alter transporter activity and cell behavior. [1]

622.143

C, 57.88; H, 4.21; N, 4.50; O, 33.41

1461-15-0

Calcein (tetraethyl ester);1170856-93-5

65079

Yellow to orange solid powder

1.7±0.1 g/cm3

952.7±65.0 °C at 760 mmHg

200 °C

530.0±34.3 °C

0.0±0.3 mmHg at 25°C

1.775

1.56

6

15

12

45

1070

0

O1C(C2=C([H])C([H])=C([H])C([H])=C2C21C1C([H])=C(C([H])([H])N(C([H])([H])C(=O)O[H])C([H])([H])C(=O)O[H])C(=C([H])C=1OC1C([H])=C(C(C([H])([H])N(C([H])([H])C(=O)O[H])C([H])([H])C(=O)O[H])=C([H])C2=1)O[H])O[H])=O

DEGAKNSWVGKMLS-UHFFFAOYSA-N

InChI=1S/C30H26N2O13/c33-21-7-23-19(5-15(21)9-31(11-25(35)36)12-26(37)38)30(18-4-2-1-3-17(18)29(43)45-30)20-6-16(22(34)8-24(20)44-23)10-32(13-27(39)40)14-28(41)42/h1-8,33-34H,9-14H2,(H,35,36)(H,37,38)(H,39,40)(H,41,42)

2-[[7'-[[bis(carboxymethyl)amino]methyl]-3',6'-dihydroxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-2'-yl]methyl-(carboxymethyl)amino]acetic acid

Fluorexon; 1461-15-0; Oftasceine; Oftasceinum; Oftasceina; 2,2',2'',2'''-(((3',6'-Dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-2',7'-diyl)bis(methylene))bis(azanetriyl))tetraacetic acid; Oftasceine [INN];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

0.1 M NaOH : ~10 mg/mL (~16.06 mM)
DMSO : ~5 mg/mL (~8.03 mM)

Solubility in Formulation 1: ≥ 0.5 mg/mL (0.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 20 mg/mL (32.13 mM) in 2% NaHCO3 in ddH2O (add these co-solvents sequentially from left to right, and one by one), clear solution; Need ultrasonic and adjust pH to 6 with NaHCO3.

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 (Please use freshly prepared in vivo formulations for optimal results.)