Calcein (mixture of isomers) is a calcium-dependent fluorescent molecule. Calcein mixture of isomers can be used to study bone metabolism (in vivo) and to stain depressed areas (in vitro). Calcein mixture of isomers can also be used for fluorometry and EDTA titration of calcium.

Physicochemical Properties

Molecular Formula	C32H34N2O13
Molecular Weight	622.53
Exact Mass	622.143
Elemental Analysis	C, 57.88; H, 4.21; N, 4.50; O, 33.41
CAS #	154071-48-4
Appearance	Yellow to orange solid powder
Density	1.7±0.1 g/cm3
Boiling Point	952.7±65.0 °C at 760 mmHg
Flash Point	530.0±34.3 °C
Vapour Pressure	0.0±0.3 mmHg at 25°C
Index of Refraction	1.775
LogP	1.56
Synonyms	154071-48-4; Calcein (mixture of isomers) [for Fluorometric Determination of Ca]; N,N'-[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-ar',ar'-diyl)bis(methylene)]bis[N-(carboxymethyl)glycine]
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Fluorescent Dye; Emission (Em) = 512; Excitation (Ex) = 488
ln Vitro	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.
Enzyme Assay	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].
Cell Assay	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. 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. 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.
References	[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.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~160.63 mM) 1M NaOH : 10 mg/mL (~16.06 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.02 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 25.0 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. Solubility in Formulation 2: 2.5 mg/mL (4.02 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: 2.5 mg/mL (4.02 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		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

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.