Fluorecent dye/Ca2+ chelator

1. Preparation of Fluo-3 AM Working Solution
1.1 Stock Solution Preparation
Dissolve Fluo-3 AM powder in anhydrous DMSO to prepare a 10 mM stock solution.
Note: The stock solution should be aliquoted and stored at -20°C or -80°C, protected from light.
1.2 Working Solution Preparation
Dilute the stock solution with pre-warmed HBSS buffer to obtain a working solution with a final concentration of 1-10 μM.
Note: Adjust the working solution concentration according to experimental needs, and prepare it fresh before use.
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2. Staining Procedure for Suspension Cells
2.1 Centrifuge to collect cells, wash twice with PBS (5 min each), and adjust cell density to 1×10⁶/mL.
2.2 Add 1 mL of working solution and incubate at room temperature in the dark for 5-30 min.
2.3 Centrifuge at 400 g for 3-4 min and remove the supernatant.
2.4 Wash cells twice with PBS (5 min each).
2.5 Resuspend cells in 1 mL HBSS and immediately analyze by fluorescence microscopy or flow cytometry.
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3. Staining Procedure for Adherent Cells
3.1 Culture cells on sterile coverslips in advance.
3.2 Remove coverslips and aspirate residual medium.
3.3 Add 100 μL working solution (ensure full coverage of the cell layer) and incubate for 5-30 min.
3.4 Remove the staining solution, wash 2-3 times with fresh medium (5 min each), and observe under a fluorescence microscope.

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The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively [1].

Solutions for the production of spontaneous Ca2+ waves within permeabilised cells [1]
Permeabilised cells were perfused with a mock intracellular solution with the following composition (mM): 100 KCl, 5 Na2ATP, 10 Na2CrP, 5.5 MgCl2, 25 HEPES, 0.05 K2EGTA, pH 7.0 (20–21 °C). The [Ca2+] in the perfusing solution was varied by the addition of known amounts of 1 M CaCl2 stock solution (BDH). An estimate of the [Ca2+] in these solutions was calculated using a computer program (React, GLSmith). For example, in the absence of added Ca2+, and assuming ∼5 μM contamination, free Ca2+ in 50 μM EGTA is ∼40 nM. Addition of Ca2+ to achieve a total [Ca2+] of ∼15 μM generates a free [Ca2+] of ∼150 nM. Further addition of Ca2+ to achieve a total of 25 μM generates a free [Ca2+] of ∼300 nM. Fluorescent Ca2+ indicators Fluo-3 (AM) or Fluo-5F were added to the solution to give a nominal final concentration of 10 μM.

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2.3. Solutions for the production of spontaneous Ca2+ waves within intact cells [1]
Intact cells were perfused with a modified Krebs solution, with the following composition (mM):140 NaCl, 4 KCl, 1 MgCl2, 5 HEPES, 11.1 Glucose, 0.3 NaH2PO4·2H2O, 0.01 Strophanthidin, pH 7.4 (20–21 °C). The [Ca2+] in the perfusing solution was varied (0.1–1 mM) by the addition of known amounts of 1 M CaCl2 stock solution (BDH). The solution used to obtain a maximal fluorescence reading where the indicator dye was saturated with Ca2+ (Fmax) contained the above solution with the addition 0.02 mM ionomycin and an increased [Ca2+] of 20 mM. To load cells with Fluo-3AM (acetoxymethyl ester form), the cardiomyocytes were incubated in 20 μM Fluo-3 (AM) for 10 min at 37 °C. The cells were centrifuged (5×g, 15 s), re-suspended in modified Krebs solution at the desired concentration of cells and left for 20 min before use. The perfusing solution used during electrical stimulation experiments was the modified Krebs described earlier with the addition of 1.8 mM CaCl2 (without strophanthidin).

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Laser-scanning confocal Fluo-3 fluorescence measurements in free solution and permeabilised cardiomyocytes [1]
The Ca2+ sensitivity of Fluo-3 and Fluo-5 in free solution was measured using a series of Ca2+ buffered solutions (10 mM EGTA) based on the mock intracellular solution described earlier. The equilibrium concentrations of metal ions in the calibration solutions were calculated using a computer program with known affinity constants for H+, Ca2+ and Mg2+ for EGTA and for ATP and CrP. Corrections for ionic strength, details of pH measurement, allowance for EGTA purity and the principles of the calculations are detailed elsewhere.

The behaviour of Fluo-3 in permeabilised cardiomyocytes was established by making simultaneous fluorescence measurements from a 6 μm (x) by 0.5 μm (y) by 0.9 μm (z) volume (20 pixels) within a myocyte and in the perfusing solution adjacent to the myocyte. In these calibration experiments, SR Ca2+ uptake was inhibited by prior treatment with thapsigargin (Calbiochem, 10 μM, 20 min). These simultaneous measurements were also used to examine Ca2+ waves inside the cells in relation to the extracellular [Ca2+], but no thapsigargin was added to these cells. Confocal line-scan images were recorded using a BioRad Radiance 2000 confocal system. Fluo-3 (or Fluo-5F) in the perfusing solution was excited at 488 nm and measured above 500 nm (HQ500LP emission filter) using epifluorescence optics of a Nikon Eclipse inverted microscope with a Fluor 60× water objective lens (Plan Apochromat NA 1.2). The 3 mW Kr laser was set to 12% power, and the gain on the photomultiplier tube (PMT) set to 25%. Iris diameter was set at 1.9 providing an axial (z) resolution of about 0.9 μm and x–y resolution of about 0.5 μm based on full width half maximal amplitude measurements of images of 0.1 μm fluorescent beads. Data was acquired in line-scan mode at 2 ms/line; pixel dimension was 0.3 μm (512 pixels/scan; zoom=1.4). The scanning laser line was oriented parallel with the long axis of the cell and placed approximately equidistant between the outer edge of the cell and the nucleus/nuclei, to ensure the nuclear area was not included in the scan line. The LaserScan software saved the data as a series of image files each containing 30,000 line-scans (i.e. 1 min of continuous recording). An experimental record typically comprised 4–5 line-scan image files; these were reviewed off-line and a single intracellular region (20 pixels wide) was selected on the basis of the earliest events in the majority of Ca2+ waves. This ensured that any movement artefact following the increase in [Ca2+] did not affect the estimation of peak [Ca2+]. The same methods were used to record and analyse the data from Ca2+ waves and stimulated transients within intact cells. Cellular autofluorescence values (Fca) were obtained by measuring intrinsic fluorescence from a cardiomyocytes not loaded with Fluo-3 or bathed in fluorescent solution. Cardiomyocytes (either intact of permeabilised) were bathed in a low (<50 nM) Ca2+ solution and imaged using the same laser power and PMT gain setting used to make the experimental measurements. Average autofluorescence values were subtracted from the fluorescence measurements to obtain accurate values of Fluo-3/Fluo-5F associated fluorescence (intact cells mean Fca=9.8±0.49 (n=4), permeabilised cells mean Fca=5.29±0.43 (n=6), extracellular Fca=3.56±0.21 (n=6)).

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Description: Fluo-3AM is a calcium indicator with green fluorescence, it can be used in calcium flux analysis.
Method: For calcium flux analysis.
1. Incubate cultured cells with Fluo-3AM (4 µM; 37°C; dark).
2. Wash cells for 3 times and re-suspended in Hank’s Balanced Salt Solution without Ca2+ and Mg2+.
3. Fluo-3AM fluorescence is assessed using the fluorescein isothiocyanate (FITC) channel and fluorescent images are visualized through confocal laser scanning microscopy.

[1]. Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics. Cell Calcium. 2003 Jul;34(1):1-9.

The purpose of this study was to establish the intracellular Kd of the commonly used Ca2+ indicator Fluo-3 (AM) in cardiomyocytes and compare the Ca2+ wave characteristics in permeabilised and intact cells. This paper shows that the permeabilised cardiomyocyte can be used as a simplified system allowing accurate indicator calibration and therefore measurement of cytosolic [Ca2+] during Ca2+ wave activity and stimulated Ca2+ transients.
This study emphasises the need to establish the behaviour of the indicator within the cytosol of the cell under study, before attributing values to the [Ca2+] from Fluo-3 fluorescence. Based on the measurements in this study, the Kd for Fluo-3 in free solution is appropriate for measurements on permeabilised cells and perhaps for Fluo-3 (AM) acid loaded into cardiomyocytes via a patch pipette. However, this Kd is not necessarily appropriate for cardiomyocytes loaded with Fluo-3 by incubation in the −AM form. [1]

C51H50CL2N2O23

1129.85

1128.218

121714-22-5

5086914

Brown to reddish brown solid powder

1.5±0.1 g/cm3

1075.8±65.0 °C at 760 mmHg

604.4±34.3 °C

0.0±0.3 mmHg at 25°C

1.631

5.4

0

25

36

78

2220

0

CC1=CC(=C(C=C1)N(CC(=O)OCOC(=O)C)CC(=O)OCOC(=O)C)OCCOC2=C(C=CC(=C2)C3=C4C=C(C(=O)C=C4OC5=CC(=C(C=C53)Cl)OCOC(=O)C)Cl)N(CC(=O)OCOC(=O)C)CC(=O)OCOC(=O)C

PTPUOMXKXCCSEN-UHFFFAOYSA-N

InChI=1S/C51H50Cl2N2O23/c1-28-7-9-39(54(19-47(62)74-24-69-30(3)57)20-48(63)75-25-70-31(4)58)45(13-28)66-11-12-67-46-14-34(8-10-40(46)55(21-49(64)76-26-71-32(5)59)22-50(65)77-27-72-33(6)60)51-35-15-37(52)41(61)17-42(35)78-43-18-44(38(53)16-36(43)51)73-23-68-29(2)56/h7-10,13-18H,11-12,19-27H2,1-6H3

acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-dichloro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-N-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate

Fluo-3AM; 121714-22-5; FLUO 3/AM; Glycine, N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-, (acetyloxy)methyl ester; Fluo-3, AM; Fluo-3-(acetoxymethyl) ester; CHEMBL2074907; SCHEMBL16364726;

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)

DMSO : ~12.5 mg/mL (~11.06 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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