Fluorecent dye/Ca2+ chelator

1. Preparation of Fluo-3FF AM Working Solution
1.1 Stock Solution Preparation
Dissolve Fluo-3FF AM in 0.025% (w/v) Pluronic F-127/DMSO solution to prepare a 1 mM stock solution.
Note: The prepared stock solution should be aliquoted and stored at -20°C or -80°C in the dark.
1.2 Working Solution Preparation
Dilute the stock solution with balanced buffer solution to prepare a 5 μM working solution.
Note: The working solution concentration can be adjusted according to experimental requirements and should be prepared fresh before use.

2. Cell Staining Procedure
2.1 Culture adherent cells on sterile coverslips in advance.
2.2 During the experiment, remove the coverslips and carefully aspirate the residual medium.
2.3 Add 100 μL of dye working solution, gently swirl to ensure complete coverage of the cell layer, then incubate for 60 minutes.
2.4 After incubation, remove the dye solution and wash the cells with 4°C pre-cooled PSS solution for 60 minutes.

Visualisation of intracellular calcium stores: To visualise the distribution of intracellular calcium stores within the myocytes, the low-affinity (Kd=42 μM) fluorescent Ca2+-sensitive indicator fluo-3FF (Abs/Em=462 nm/526 nm) was used. This dye was selected among other low-affinity Ca2+-sensitive indicators because of its insensitivity to Mg2+ and relatively high photostability. The myocytes were loaded with fluo-3FF by exposure to 5 μM Fluo-3FF AM (diluted from a stock containing 1 mM Fluo-3FF AM and 0.025% (w/v) pluronic F-127 in dimethyl sulphoxide) for 60–90 min at room temperature, followed by 60-min wash in PSS at 4 °C.

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To visualise three-dimensional (3-D) distribution of the intracellular calcium stores within the cell, z-sectioning protocol (series of x–y images taken at defined intervals along the z-axis) was applied to the myocytes preloaded with the low-affinity Ca2+-sensitive indicator Fluo-3FF (see above). This protocol was comprised of 30–45 individual x–y images each taken from a confocal optical section below 0.8 μm with a z-step of 0.4 μm. Fluo-3FF fluorescence was excited by the 488 nm line of a 200 mW argon ion laser and the emitted fluorescence was detected at wavelengths above 505 nm [1].

Intracellular calcium stores of human uterine myocytes in primary and second passage cell culture were visualized using the low-affinity calcium-sensitive fluorescent dye, fluo-3FF. The calcium stores appeared as numerous small (0.2-0.5 microm diameter) focal fluorescences. The stores were not depleted by exposing the cells to oxytocin or ryanodine under standard conditions. The stores were rapidly depleted by oxytocin or ryanodine exposure when sarcoplasmic reticulum (SR) calcium re-uptake was inhibited by pretreatment with thapsigargin. Immunofluorescence experiments indicated that both ryanodine and inositol 1,4,5-trisphosphate (IP(3)) receptors were smoothly distributed throughout the SR, and neither receptor co-localized with the calcium stores. Since IP(3) and ryanodine calcium channels are tightly associated with their receptor, these results suggest that SR calcium release occurs via second messenger channels that are remote from the SR calcium stores. These observations are consistent only with a mechanism for release of calcium stores where the SR serves three functions: (1) as site of calcium storage, (2) as the structure that contains the IP(3)- and ryanodine receptors and their associated release channels, and (3) as a conduit between the calcium stores and the release channels. [2]

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The dynamics of carbachol (CCh)-induced [Ca(2+)](i) changes was related to the kinetics of muscarinic cationic current (mI(cat)) and the effect of Ca(2+) release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on mI(cat) was evaluated by fast x-y or line-scan confocal imaging of [Ca(2+)](i) combined with simultaneous recording of mI(cat) under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca(2+)-sensitive indicator fluo-3, x-y confocal imaging revealed CCh (10 microM)-induced Ca(2+) waves, which propagated from the cell ends towards the myocyte centre at 45.9 +/- 8.8 microms(-1) (n = 13). Initiation of the Ca(2+) wave preceded the appearance of any measurable mI(cat) by 229 +/- 55 ms (n = 7). Furthermore, CCh-induced [Ca(2+)](i) transients peaked 1.22 +/- 0.11s (n = 17) before mI(cat) reached peak amplitude. At -50 mV, spontaneous release of Ca(2+) through RyRs, resulting in Ca(2+) sparks, had no effect on CCh-induced mI(cat) but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca(2+) release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI(cat) (n = 14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI(cat) when [Ca(2+)](i) was strongly buffered with Ca(2+)/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca(2+) sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca(2+) upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca(2+) potentiated mI(cat) in the same way as in control). In contrast, IP(3)R-mediated Ca(2+) release upon flash photolysis of "caged" IP(3) (30 microM in the pipette solution) augmented mI(cat) (n = 15), even though [Ca(2+)](i) did not reach the level required for potentiation of mI(cat) during photorelease of Ca(2+) (n = 10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca(2+) indicator Fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP(3)R type 1 and to RyRs revealed that IP(3)Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP(3)R-mediated Ca(2+) release plays a central role in the modulation of mI(cat) in the guinea-pig ileum and that IP(3) may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca(2+) [1].

[1]. Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca2+ release: a central role of inositol 1,4,5-trisphosphate receptors. Cell Calcium. 2004 Nov;36(5):367-86.

[2]. Focal sarcoplasmic reticulum calcium stores and diffuse inositol 1,4,5-trisphosphate and ryanodine receptors in human myometrium. Cell Calcium. 1999 Jul-Aug;26(1-2):69-75.

The tight coupling between IP3-induced Ca2+ release (IICR) and muscarinic cation channel gating suggests that sarcoplasmic reticulum (SR) elements in ileal muscle cells are located submembrane-bound. To observe the spatial arrangement of intracellular calcium stores in live smooth muscle cells (SMCs) freshly isolated from the longitudinal lamina of guinea pig ileum, we used the low-affinity Ca2+-sensitive indicator fluo-3FF. Myocytes were incubated with 5 μM Fluo-3FF AM for 60–90 min, followed by washing with PSS buffer for 60 min to deesterify the indicator (see Section 2.3), thereby loading the indicator into the cells. Due to the low affinity of this dye for Ca2+ (Kd = 42 μM), the fluorescence signal in the intracellular [Ca2+]i < 1 μM region was weak and removed from the images by thresholding, thus preventing imaging of cytoplasmic Ca2+. To visualize the three-dimensional distribution of intracellular calcium store components, we employed z-axis slicing (see Section 2.5). This technique involves acquiring 35–40 fluorescence images in the xy direction from confocal optical sections (<0.8 μm) of myocytes, with a z-axis step size (objective displacement) of 0.4 μm (n=12). This experimental method revealed that the calcium reservoir within ileal myocytes consists of a well-developed subplasmic sarcoplasmic reticulum and some perinuclear structures (likely the nuclear membrane and Golgi apparatus), which appear to be connected to the surface sarcoplasmic reticulum (Fig. 8A). The distribution of calcium reservoir components within ileal myocytes is largely similar to our recent observations using DiOC6 and BODIPY TR-X rhinodrine in rabbit portal vein myocytes. As shown in Fig. 8B, the arrangement pattern of the intracellular calcium reservoir was further confirmed by xy imaging fluo-3FF fluorescence scanning (confocal optical sections <0.8 μm) of 35 ileal myocytes. The data suggests that the sarcoplasmic reticulum in ileal myocytes is well-developed in the submembrane position and may transport Ca2+ ions with high precision in time and space, thereby regulating Ca2+-dependent ion channels on the plasma membrane [1].

C50H46CL2F2N2O23

1151.80266141891

1150.183

348079-13-0

3626995

Typically exists as solid at room temperature

1.5±0.1 g/cm3

1087.3±65.0 °C at 760 mmHg

611.4±34.3 °C

0.0±0.3 mmHg at 25°C

1.611

4.94

0

27

36

79

2270

0

ClC1C(=CC2=C(C=1)C(=C1C=C(C(C=C1O2)=O)Cl)C1C=CC(=C(C=1)OCCOC1C(=C(C=CC=1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)F)F)N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)OCOC(C)=O

ABZBPPQDKRLSCZ-UHFFFAOYSA-N

InChI=1S/C50H46Cl2F2N2O23/c1-26(57)69-21-74-42-16-41-33(14-35(42)52)48(32-13-34(51)39(62)15-40(32)79-41)31-6-8-37(55(17-44(63)75-22-70-27(2)58)18-45(64)76-23-71-28(3)59)43(12-31)67-10-11-68-50-38(9-7-36(53)49(50)54)56(19-46(65)77-24-72-29(4)60)20-47(66)78-25-73-30(5)61/h6-9,12-16H,10-11,17-25H2,1-5H3

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

Fluo-3FF AM; 348079-13-0; acetyloxymethyl 2-[4-[3-(acetyloxymethoxy)-2,7-dichloro-6-oxoxanthen-9-yl]-N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[6-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]-2,3-difluorophenoxy]ethoxy]anilino]acetate; N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[6-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-2,3-difluorophenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-glycine(acetyloxy)methylester; HY-D1755; N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[6-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-2,3-difluorophenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-glycine(acetyloxy)methyl ester; PD044414;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)