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LDS-751 Sale

目录号 : GC30134

LDS-751是一种核酸染色剂。

LDS-751 Chemical Structure

Cas No.:181885-68-7

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5mg
¥1,035.00
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10mg
¥1,845.00
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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

Cell experiment:

Blood samples (1 mL) are obtained using a sterile Butterfly-21 needle and plastic syringe from the antecubital vein of normal healthy volunteers who have given their informed consent. They are immediately transferred to plastic tubes containing 17.3 mg of phenylmethylsulphonyl fluoride (PMSF) and 1 mL of LDS-751 at room temperature. Aliquots (25 μL) are incubated for 5 min with between 2 μL and 5 μL of undiluted monoclonal antibodies, then diluted with 0.5 mL of 1% BSA in Hepesbuffered Hanks' balanced salts solution (HHBSS) and examined by flow cytometry[2].

References:

[1]. Terstappen LW, et al. A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations. J Immunol Methods. 1989 Sep 29;123(1):103-12.
[2]. McCarthy DA, et al. A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood. J Immunol Methods. 1993 Aug 9;163(2):155-60.

产品描述

LDS-751 is a nucleic acid stain.

The LDS-751 permits the discrimination of intact cells from residual erythrocyte ghosts, platelets and damaged nucleated cells. The forward and orthogonal fight scattering signals of the intact cells, identified by LDS-751 staining allow a clear separation between the major leukocyte populations since the damaged nucleated cells, erythrocytes, erythrocyte cell ghosts and platelets are removed from the display[1].

[1]. Terstappen LW, et al. A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations. J Immunol Methods. 1989 Sep 29;123(1):103-12. [2]. McCarthy DA, et al. A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood. J Immunol Methods. 1993 Aug 9;163(2):155-60.

Chemical Properties

Cas No. 181885-68-7 SDF
Canonical SMILES CC[N+]1=C2C=CC(N(C)C)=CC2=CC=C1/C=C/C=C/C3=CC=C(N(C)C)C=C3.O=Cl(=O)([O-])=O
分子式 C25H30ClN3O4 分子量 471.98
溶解度 Soluble in DMSO 储存条件 Store at -20°C,unstable in solution, ready to use.
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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。
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溶解性数据

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1 mg 5 mg 10 mg
1 mM 2.1187 mL 10.5937 mL 21.1873 mL
5 mM 0.4237 mL 2.1187 mL 4.2375 mL
10 mM 0.2119 mL 1.0594 mL 2.1187 mL
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Research Update

Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein

Eur J Biochem 1998 May 15;254(1):181-8.9652412 10.1046/j.1432-1327.1998.2540181.x

P-glycoprotein is an ATP-dependent transporter of an extremely wide variety of lipophilic compounds. We showed previously [Shapiro, A. B. & Ling, V. (1997a) Eur. J. Biochem. 250, 130-137] that P-glycoprotein contains two drug transporting sites, dubbed H (for Hoechst 33342-selective) and R (for rhodamine-123-selective), that interact with positive cooperativity. The H site transports 2-[2-(4-ethoxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)be nzimidazole (Hoechst 33342) from the cytoplasmic leaflet of the plasma membrane to the aqueous extracellular medium [Shapiro, A. B. & Ling, V. (1997b) Eur. J. Biochem. 250, 122-129]. The environment from which the R site transports its substrates is unknown. In this paper, we used the fluorescent DNA dye 2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-3-ethylbenzothiazolium perchlorate (LDS-751), a substrate of the R site, to address this issue. LDS-751 which, like Hoechst 33342, exhibits lipid-dependent fluorescence and slow transleaflet diffusion, allowed us to use the same methodology that we used for the H site to study the location of the R site. As with Hoechst 33342, the specific initial rate of LDS-751 transport by P-glycoprotein-rich, isolated plasma membrane vesicles from CH(R)B30 cells was directly proportional to the amount of membrane-bound LDS-751 and inversely proportional to the concentration of free, aqueous LDS-751. This result demonstrates that the R site of P-glycoprotein transports LDS-751 out of the lipid membrane. The slight decrease, instead of an increase, in the initial rate of active transport of LDS-751 with the amount of time elapsed for slow diffusion of LDS-751 from the cytoplasmic leaflet to the extracellular leaflet indicates that the R site of P-glycoprotein removes LDS-751 from the cytoplasmic leaflet of the plasma membrane. Thus, both known drug-transporting sites of P-glycoprotein remove their substrates from the cytoplasmic leaflet. Since all of the P-glycoprotein substrates we have examined so far are recognized by one or both of the two known drug-transporting sites, these two sites in the cytoplasmic leaflet of the plasma membrane may be able to account for all substrate transport by P-glycoprotein.

Staining of cellular mitochondria with LDS-751

J Immunol Methods 2001 Nov 1;257(1-2):35-40.11687236 10.1016/s0022-1759(01)00440-9

We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02-20 microg/ml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing.

Interaction of LDS-751 with the drug-binding site of P-glycoprotein: a Trp fluorescence steady-state and lifetime study

Arch Biochem Biophys 2009 Dec;492(1-2):17-28.19818729 10.1016/j.abb.2009.10.002

P-glycoprotein (ABCB1) is an ATP-driven efflux pump which binds drugs within a large flexible binding pocket. Intrinsic Trp fluorescence was used to probe the interactions of LDS-751 (2-[4-(4-[dimethylamino]phenyl)-1,3-butadienyl]-3-ethylbenzo-thiazolium perchlorate) with purified P-glycoprotein, using steady-state/lifetime measurements and collisional quenching. The fast decay component of P-glycoprotein intrinsic fluorescence (tau(1)=0.97 ns) was unaffected by LDS-751 binding, while the slow decay component (tau(2)=4.02 ns) was quenched by dynamic and static mechanisms. Both the wavelength-dependence of the decay kinetics, and the time-resolved emission spectra, suggested the existence of excited-state relaxation processes within the protein matrix on the nanosecond time-scale, which were altered by LDS-751 binding. The fast decay component, which is more solvent-exposed, can be attributed to cytosolic/extracellular Trp residues, while the slow decay component likely arises from more buried transmembrane Trp residues. Interaction of a drug with the binding pocket of P-glycoprotein thus affects its molecular structure and fast dynamics.

Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs

Biochemistry 2005 Oct 25;44(42):14020-9.16229491 10.1021/bi0511179

The P-glycoprotein efflux pump, an ABC superfamily member, can export a wide variety of hydrophobic drugs, natural products, and peptides from cells, powered by the energy of ATP hydrolysis. Transport substrates appear to first partition into the membrane and then interact with the protein within the cytoplasmic leaflet. Two drug binding sites within P-glycoprotein have been described which interact allosterically, the H-site (binds Hoechst 33342) and the R-site (binds rhodamine 123); however, the structural and functional relationship between the various binding sites appears complex. In this work, we have used fluorescence spectroscopic approaches to characterize the interaction of the transporter with LDS-751 and rhodamine 123, both of which are believed to bind to the putative R-site based on functional transport studies. By carrying out single and sequential dual fluorescence titrations of purified P-glycoprotein with the two substrates, we observed that bound LDS-751 interacted with bound rhodamine 123. Rhodamine 123 and LDS-751 showed a reciprocal negative interaction, each reducing the binding affinity of the other by 5-fold, indicating that the two compounds were simultaneously bound to the protein to form a ternary complex. Fitting of the dependence of the apparent Kd for LDS-751 binding on rhodamine 123 concentration suggested that the two compounds interacted noncompetitively. We conclude that the two-site drug binding model for P-glycoprotein requires modification. The putative R-site appears large enough to accommodate two compounds simultaneously. The locations where LDS-751 and rhodamine 123 bind are likely adjacent to each other, possibly overlapping, and may be within a hydrophobic pocket.

Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site

Biochemistry 2005 Jan 18;44(2):643-55.15641790 10.1021/bi0485326

One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 microM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites. The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane.