Phalloidin   中文名称: 鬼笔鹅膏素

Fluorescent Dye; F-actin

鬼笔环肽染色对固定在含有 0.2% 戊二醛的 PBS 中的细胞效果良好，但对固定细胞则效果不佳（可能是因为肌动蛋白丝断裂的缺陷）[1]。

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真核细胞已经进化到划分其功能并在细胞间运输各种代谢物。因此，在细胞生物学中，研究组织和结构/功能关系具有重要意义。细胞骨架由一系列丝状结构组成，包括中间丝、肌动蛋白丝和微管。免疫荧光染色最常用于研究细胞骨架成分。然而，也可以用荧光标记分离的细胞骨架蛋白，并将其显微注射回细胞中，或将其添加到固定的、可渗透的细胞中。或者，如本文所述，可以使用蘑菇衍生的荧光毒素，phalloidin或phallacidin来标记细胞骨架的F-actin。Phalloidin可用不同的荧光团标记。特定荧光团的选择应取决于生物碱标记肌动蛋白是否是双重标记实验的一部分。在大多数细胞中，肌动蛋白丝的丰富应该提供非常强烈的信号。在双标记实验中，应选择荧光团来考虑这一点。一般来说，罗丹明标签更耐光漂白，可以承受更精细结构所需的更长时间的曝光[1]。

1. 用PBS将荧光标记的鬼笔环肽（300单位/mL原液）按1:200比例稀释 [1]
2. 吸弃培养于盖玻片上的细胞培养基
3. 用PBS漂洗细胞三次
4. 用3.7%甲醛溶液固定细胞10分钟
5. PBS漂洗已固定细胞三次（每次5分钟）
6. 用0.2% Triton X-100溶液透化处理细胞5分钟
7. PBS再次漂洗三次
8. 室温下用荧光标记鬼笔环肽染色5-10分钟
9. PBS漂洗三次（每次5分钟）
10. 封片后根据所用探针选择相应荧光滤光片观察
注：所有操作步骤均需使用无菌PBS缓冲液完成。

Absorption, Distribution and Excretion
THE UPTAKE, BINDING AND ELIMINATION OF PHALLOIDIN BY LIVER WAS COMPARED IN ADULT AND BABY (17-19 DAYS OLD) RATS IN VIVO AND IN VITRO. IN BOTH GROUPS, THERE WAS NO RELATION BETWEEN THE CONCENTRATION OF THE POISON IN THE LIVER AND THE TOXICITY. ALTHOUGH BABY RATS SHOWED A SIGNIFICANTLY HIGHER TOLERANCE AGAINST PHALLOIDIN THAN THE ADULT ANIMALS, THE CONCENTRATION OF THE POISON IN THE LIVER OF BABY RATS WAS HIGHER, AND THE ELIMINATION WAS SIGNIFICANTLY SLOWER THAN IN ADULT RATS. THE VERY TIGHT BINDING AND CONCENTRATION OF PHALLOIDIN IN THE LIVER WAS EXPLAINED BY AN EXTREMELY LOW DISSOCIATION CONSTANT.
PHALLOIN AND GAMMA-AMANITIN WERE DETECTED WITHIN THE FIRST DAY AND PHALLOIDIN WITHIN THE SECOND DAY IN THE URINE OF RATS AFTER ADMINISTRATION OF AMANITA PHALLOIDES EXTRACTS.

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Metabolism / Metabolites
VARIOUS OBSERVATIONS SUGGESTED THAT PHALLOIDINE, A POISONOUS CONSTITUENT OF THE GREEN MUSHROOM, AMANITA PHALLOIDES, WOULD NOT BE TOXIC PER SE, BUT WOULD UNDERGO BIOACTIVATION IN THE LIVER. USING (3)H-DEMETHYLPHALLOIN, A TOXIC DERIVATIVE OF PHALLOIDINE, /IT WAS CONCLUDED/...THAT (3)H-DEMETHYLPHALLOIN, AND MOST PROBABLY ALSO PHALLOIDINE, ARE UNMETABOLIZED IN THE LIVERS OF RATS AND MICE. A SECONDARY EXCRETORY PRODUCT IN THE URINE OF TREATED ANIMALS WAS FOUND TO BE AN AUTODEGRADATION COMPOUND OF THE (3)H-LABELED DERIVATIVE.

Interactions
PRETREATMENT WITH RIFAMPICIN (100 OR 300 MG/KG, ORAL) PROTECTED MICE AGAINST THE TOXICITY OF PHALLOIDIN (3 MG/KG, IP) AS REFLECTED BY THE DECREASE IN THE MORTALITY RATE.
IN MICE, THE ACUTE HEMORRHAGIC NECROSIS OF THE LIVER INDUCED BY PHALLOIDIN WAS PREVENTED BY TREATMENT WITH SILYBIN, A PLANT COMPOUND ISOLATED FROM SILYBUM MARIANUM. PRETREATMENT WITH A SINGLE DOSE OF SILYBIN COMPLETELY ABOLISHED THE MORPHOLOGIC CHANGES INDUCED BY THE TOXIN, AND SIGNIFICANTLY DECREASED THE ACTIVITIES OF SERUM ENZYMES.

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Toxicity Summary
It binds actin, preventing its depolymerization and poisoning the cell. Phalloidin binds specifically at the interface between F-actin subunits, locking adjacent subunits together. Phalloidin, a bicyclic heptapeptide, binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the rate constant for the dissociation of actin subunits from filament ends, which essentially stabilizes actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin (L1183).

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LD50: 2 mg/kg (Mouse) (T258)

[1]. Chazotte B. Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging. Cold Spring Harb Protoc. 2010 May;2010(5):pdb.prot4947.

Phalloidin is a homodetic bicyclic heptapeptide having a sulfide bridge.
Phalloidine has been reported in Amanita suballiacea, Amanita phalloides, and other organisms with data available.
Very toxic polypeptide isolated mainly from AMANITA phalloides (Agaricaceae) or death cup; causes fatal liver, kidney and CNS damage in mushroom poisoning; used in the study of liver damage.

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Mechanism of Action
IN CONTRAST TO NORMAL LIVER CELLS, AS-30D RAT HEPATOMA CELLS ARE INSENSITIVE TO PHALLOIDIN. HEPATOMA CELLS APPARENTLY DO NOT CONSUME THE TOXIN AS DO NORMAL LIVER CELLS.
TOLERATED DOSES OF PHALLOIDIN PROTECT MICE AGAINST LETHAL DOSES OF PHALLOIDIN. RESISTANCE IS CONFERRED BY THE 1/10 LD95 OF PHALLOIDIN AND SETS IN AT ABOUT 8 HOURS AFTER PRETREATMENT.
WITHIN 1 HOUR OF EXPOSURE OF PRIMARY CULTURES OF ADULT RAT HEPATOCYTES TO PHALLOIDIN AT 50 MUG/ML, 60-70% OF THE CELLS WERE DEAD (TRYPAN BLUE-STAINABLE). THERE WAS NO LOSS OF VIABILITY OF THE SAME CELLS EXPOSED TO PHALLOIDIN IN CULTURE MEDIUM DEVOID OF CA2+. INITIALLY PHALLOIDIN INTERACTS IN A CA2+-INDEPENDENT PROCESS WITH CELL MEMBRANE-ASSOCIATED ACTIN. THE 2ND STEP IS A CA2+-DEPENDENT PROCESS THAT MOST LIKELY REPRESENTS AN INCREASED INFLUX OF CA2+ ACROSS A COMPROMISED CELL MEMBRANE PERMEABILITY BARRIER AND DOWN THE STEEP CONCENTRATION GRADIENT THAT EXISTS BETWEEN THE OUTSIDE AND INSIDE OF THE CELL. THESE RESULTS STRENGTHEN THE HYPOTHESIS THAT DISTURBANCES IN CA2+ HOMEOSTASIS ARE INDUCED IN VIVO.
PHALLOIDIN ADMINISTERED TO MALE RATS FOR 7 DAYS (500 MUG/KG/DAY) INCREASED THE MEAN HEPATIC CONTENT OF FILAMENTOUS ACTIN. BOTH BILE FLOW AND BILE ACID EXCRETION DIMINISHED PROPORTIONALLY. MICROFILAMENTS MAY INFLUENCE THE PERMEABILITY OF TIGHT JUNCTIONS BETWEEN HEPATOCYTES. BILE CONSTITUENTS MIGHT REFLUX FROM THE CANALICULUS TO THE INTERCELLULAR SPACE IN PHALLOIDIN-INDUCED CHOLESTASIS.

C35H48N8O12S

804.867020000001

788.316

17466-45-4

441542

White to off-white solid powder

1.51g/cm3

1370.5ºC at 760mmHg

MAXIMUM ABSORPTION (WATER): 295 NM (E= 0.597, 1%, 1 CM); MELTING POINT: 280-282 °C /HEXAHYDRATE/

782.6ºC

0mmHg at 25°C

1.683

-1.7

316.92

11

12

4

55

1510

10

C[C@H]1C(=O)N[C@H]2CC3=C(NC4=CC=CC=C34)SC[C@@H](C(=O)N5C[C@H](C[C@H]5C(=O)N1)O)NC(=O)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](NC2=O)C[C@](C)(CO)O)C)[C@H](C)O

KPKZJLCSROULON-QKGLWVMZSA-N

InChI=1S/C35H48N8O11S/c1-15-27(47)38-22-10-20-19-7-5-6-8-21(19)41-33(20)55-13-24(34(53)43-12-18(46)9-25(43)31(51)37-15)40-32(52)26(17(3)45)42-28(48)16(2)36-30(50)23(39-29(22)49)11-35(4,54)14-44/h5-8,15-18,22-26,41,44-46,54H,9-14H2,1-4H3,(H,36,50)(H,37,51)(H,38,47)(H,39,49)(H,40,52)(H,42,48)/t15-,16-,17-,18-,22-,23-,24-,25-,26+,35+/m0/s1

(1S,14R,18S,20S,23S,28S,31S,34R)-28-[(2R)-2,3-dihydroxy-2-methylpropyl]-18-hydroxy-34-[(1S)-1-hydroxyethyl]-23,31-dimethyl-12-thia-10,16,22,25,27,30,33,36-octazapentacyclo[12.11.11.03,11.04,9.016,20]hexatriaconta-3(11),4,6,8-tetraene-15,21,24,26,29,32,35-heptone

17466-45-4; Phalloidin from Amanita phalloides; 28-(2,3-Dihydroxy-2-methylpropyl)-18-hydroxy-34-(1-hydroxyethyl)-23,31-dimethyl-12-thia-10,16,22,25,27,30,33,36-octazapentacyclo[12.11.11.03,11.04,9.016,20]hexatriaconta-3(11),4,6,8-tetraene-15,21,24,26,29,32,35-heptone; SCHEMBL39329; GTPL4736; KPKZJLCSROULON-UHFFFAOYSA-N; NSC523214; SMP1_000234;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。