TRITC Phalloidin
目录号 : GC18292
TRITC是一种橙红色荧光探针，Phalloidin，一种源自鹅膏的环状七肽毒素
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1、制备染色液
（1）染料储存液: 取低温保存的染料储存液置于室温回温至少20min，低速离心将产品集中在管底，按照单次用量对储存液进行分装，置于-20℃避光保存，一年稳定；
（或）取低温保存的冻干粉置于室温回温至少20min，低速离心后，加入甲醇或无水DMSO充分溶解配制成10-100μM储存液。配置好的储存液分装后于-20或-80℃避光保存；
（2）染料工作液: 溶液形式的产品以1000xDMSO储存液形式提供，建议使用PBS (本方案使用的PBS均为pH7.4的1xPBS)按照1:1000的比例稀释储存液，将其稀释到1x染色工作液，用枪吹打混匀；如为自行配置的染料储存液，建议使用PBS稀释成10-200nM的工作液。
注意:
① 请根据实际情况调整及优化工作液浓度，现用现配。
② 可使用含1% BSA的PBS稀释储存液，能够降低非特异背景染色，也能最小化鬼笔环肽粘附到管壁的可能性。
2、细胞染色
（1）细胞爬片培养，生长达到70-80%的汇合度。
（2）吸弃培养液，用37°C预热的PBS清洗细胞2次。
（3）使用溶于PBS的4%多聚甲醛固定细胞，室温固定10min。
注意:
① 固定过程中甲醇能破坏肌动蛋白，建议使用无甲醇的固定液；
② 也可以在含3-4%福尔马林的PBS中室温固定细胞10-30分钟。
（4）室温下使用PBS清洗细胞2-3次，30s/次。
可选步骤①：用含10 mM乙醇胺（或0.1 M甘氨酸）的PBS处理细胞5分钟，用于淬灭过量的福尔马林；
可选步骤②：将含0.1% Triton X-100的PBS添加到固定细胞中，静置3-5分钟，以增加渗透性，然后用PBS洗涤细胞2-3次。
（5）取约100μl现配的染色工作液，使其完全覆盖盖玻片上的细胞，室温避光孵育30min。
注意:
①通常情况下4℃-37℃均适合用于染色。为了避免工作液的挥发，孵育过程中将盖玻片置于一密封的容器内；
②若有需要，此时可加入不同于TRITC荧光光谱的细胞核染色液。
（6）室温下使用PBS清洗细胞2-3次，30s/次。
（7）使用滤纸尽量吸干细胞表面液体，将盖玻片倒置在滴有一滴抗荧光淬灭剂的载玻片上，用纸巾轻轻吸掉多余淬灭剂，然后用透明指甲油密封盖玻片四周。将此法处理玻片置于4℃避光存放至少6个月仍能维持F-actin染色。
（8）荧光显微镜下观察染色结果，TRITC的最大激发光/发射光为546/575nm。
注意事项：
① 可在细胞培养板/共聚皿中对细胞进行染色，步骤（7）改为滴加抗荧光淬灭剂到孔内保护荧光；
② 鬼笔环肽染色不适用于甲醇或丙酮固定的细胞，这类固定剂会破坏肌动蛋白的结构，并阻止鬼笔环肽进行染色，推荐使用0.2%戊二醛固定细胞；
③ 鬼笔环肽对pH值敏感：如果pH值升高，鬼笔环肽中关键的硫醚桥键会被裂解，从而失去对肌动蛋白的亲和力；
④ 鬼笔环肽染色可以与抗体染色搭配使用，推荐在一抗或二抗孵育期间加入鬼笔环肽偶联物；
⑤ 鬼笔环肽偶联物的最佳浓度和孵育时间取决于具体的细胞类型、固定/样本制备条件和/或细胞/组织对探针的通透性；
⑥ 悬浮细胞可以附着在多聚-D-赖氨酸微孔板或盖玻片上，然后使用贴壁细胞方案进行染色；
⑦ 如果细胞状态差，建议在染色液和洗涤液中加入血清（2-10%）；
⑧ 在某些情况下，可以使用一步法快速进行鬼笔环肽染色：4℃下，在3.7% 福尔马林和50-100 µg/mL棕榈酰溶血磷脂酰胆碱与鬼笔环肽偶联物中孵育 20分钟，然后洗涤3次并封固；
⑨ 在未固定的样本中进行染色时，鬼笔环肽的结合会降低肌动蛋白亚基与肌动蛋白丝末端的解离速率，从而通过防止肌动蛋白丝解聚来稳定肌动蛋白丝；
⑩ 对于其他样本类型的染色流程，为优化固定条件，便于染色，建议在一定范围内变更固定时间和福尔马林浓度；
⑪ 鬼笔环肽可用于经福尔马林固定和透化的组织切片、细胞培养物等样本类型以及无细胞实验，也可用于已脱蜡的石蜡包埋样本，且鬼笔环肽染色不需要抗原修复；
⑫ 鬼笔环肽的半数致死量LD50为2 mg/kg，使用时请注意防护。但通常情况下，鬼笔环肽的用量很小，不会造成重大安全风险；
⑬ 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭；
为了您的安全和健康，请穿实验服并戴一次性手套操作。
TRITC is an orange-red fluorescent probe，Phalloidin, a cyclic heptapeptide toxin derived from Amanita phalloides, selectively binds filamentous actin F-actin with high affinity (Kd= 20 nM), but not monomeric actin g-actin[1 2]. TRITC Phalloidin staining has strong specificity and high contrast. TRITC Phalloidin is widely used to immobilize permeable cells, but it can also enter living cells..
TRITC Phalloidin staining showed that MCF-7 cells transformed from round to irregular polygon with stiffness increase either in normoxia or hypoxia[5]. Cytoskeletal analysis by TRITC Phalloidin staining show that HeLa cells in the mixed hydrogel beads closely link to each other[6].The cDNAs coding for IEF's 8118(human homolog of bovine GDI ) and 8120(a distinct although related protein) were recombined into vaccinia virus and expressed in differentiated human keratinocytes and their effect on the actin cytoskeleton was assessed by immunofluorescence using TRITC Phalloidin. The results showed that overexpression of both GDI proteins leads to rounding up of the cells and loss of stress fibers and focal contact sites[4].Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine elastic modulus. The underlying changes in cytoskeleton were studied by staining the cells with TRITC Phalloidin. With estradiol treatment, elastic modulus of osteoblasts significantly decreased by 43-46%[7]
In Amoeba proteus (strain B),When used TRITC Phalloidin staining to examine the presence of F-actin in the amoeba nucleus. No significant amount of F-actin was detected in the nucleus; instead, F-actin formed a cytoplasmic meshwork of filaments and bundles supporting the nucleus[3]
References:
[1]: Bereiter-Hahn J, Kajstura J. Scanning microfluorometric measurement of TRITC-phalloidin labelled F-actin. Dependence of F-actin content on density of normal and transformed cells. Histochemistry. 1988;90(4):271-6. doi: 10.1007/BF00495970. PMID: 3230049.
[2]: Cano ML, Cassimeris L, et,al. Characterization of tetramethylrhodaminyl-phalloidin binding to cellular F-actin. Cell Motil Cytoskeleton. 1992;21(2):147-58. doi: 10.1002/cm.970210208. PMID: 1559266.
[3]: Berdieva M, Bogolyubov D, et,al. Nucleus-associated actin in Amoeba proteus. Eur J Protistol. 2016 Oct;56:191-199. doi: 10.1016/j.ejop.2016.09.002. Epub 2016 Sep 9. PMID: 27684042.
[4]: Leffers H, Nielsen MS, et,al. Identification of two human Rho GDP dissociation inhibitor proteins whose overexpression leads to disruption of the actin cytoskeleton. Exp Cell Res. 1993 Dec;209(2):165-74. doi: 10.1006/excr.1993.1298. PMID: 8262133.
[5]: Lv Y, Chen C, Zhao B, et,al. Regulation of matrix stiffness on the epithelial-mesenchymal transition of breast cancer cells under hypoxia environment. Naturwissenschaften. 2017 Jun;104(5-6):38. doi: 10.1007/s00114-017-1461-9. Epub 2017 Apr 5. PMID: 28382476.
[6]: Wang Y, Wang J. Mixed hydrogel bead-based tumor spheroid formation and anticancer drug testing. Analyst. 2014 May 21;139(10):2449-58. doi: 10.1039/c4an00015c. PMID: 24699505.
[7]: Muthukumaran P, Lim CT, et,al. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes. Biochem Biophys Res Commun. 2012 Jul 6;423(3):503-8. doi: 10.1016/j.bbrc.2012.05.149. Epub 2012 Jun 5. PMID: 22683634.
TRITC 是一种橙红色荧光探针，Phalloidin 是一种源自鹅膏菌的环状七肽毒素，可以高亲和力（Kd= 20 nM）选择性结合丝状肌动蛋白 F-肌动蛋白，但不结合单体肌动蛋白 g-肌动蛋白 [1 2]。 TRITC 鬼笔环肽染色具有很强的特异性和高对比度。 TRITC Phalloidin 广泛用于固定可渗透细胞，但它也可以进入活细胞..
TRITC Phalloidin 染色显示，MCF-7 细胞在常氧或缺氧条件下从圆形变为不规则多边形，硬度增加[5]。通过 TRITC 鬼笔环肽染色进行的细胞骨架分析表明，混合水凝胶珠中的 HeLa 细胞彼此紧密相连[6]。编码 IEF 的 8118（牛 GDI 的人类同源物）和 8120（一种独特但不同的基因）的 cDNA相关蛋白）重组为痘苗病毒并在分化的人角质形成细胞中表达，并使用 TRITC 鬼笔环肽通过免疫荧光评估它们对肌动蛋白细胞骨架的影响。结果表明，两种 GDI 蛋白的过度表达会导致细胞聚集，应力纤维和局灶性接触位点丢失[4]。已知雌激素对骨形成成骨细胞和骨有直接影响吸收破骨细胞。将细胞在单独的培养基或补充有 β-雌二醇的培养基中培养，然后进行原子力显微镜压痕 (AFM) 以确定弹性模量。通过用 TRITC 鬼笔环肽染色细胞来研究细胞骨架的潜在变化。雌二醇治疗成骨细胞弹性模量显着降低43-46%[7]
在变形虫（B 株）中，当使用 TRITC 鬼笔环肽染色检查变形虫细胞核中是否存在 F-肌动蛋白时。在细胞核中未检测到大量 F-肌动蛋白；相反，F-肌动蛋白形成了支持细胞核的细丝和束的细胞质网络[3]
| Cas No. | SDF | ||
| 分子式 | C60H70N12O13S2 | 分子量 | 1231.4 |
| 溶解度 | Soluble in DMSO, DMF, methanol or acetonitrile (20%) | 储存条件 | Store at -20°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 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。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.8121 mL | 4.0604 mL | 8.1208 mL |
| 5 mM | 0.1624 mL | 0.8121 mL | 1.6242 mL |
| 10 mM | 0.0812 mL | 0.406 mL | 0.8121 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
TRITC-Loaded PLGA Nanoparticles as Drug Delivery Carriers in Mouse Oocytes and Embryos
ACS Appl Mater Interfaces2021 Feb 10;13(5):5975-5988.PMID: 33502166DOI: 10.1021/acsami.0c19792
The structural layers around oocytes make it difficult to deliver drugs aimed at treating infertility. In this study, we sought to identify nanoparticles (NPs) that could easily pass through zona pellucida (ZP), a special layer around oocytes, for use as a drug delivery carrier. Three types of NPs were tested: quantum dot NPs, PE-polyethylene glycol (PEG)-loaded poly(lactic-co-glycolic acid) (PLGA) NPs (PEG/PL), and tetramethylrhodamine-loaded PLGA NPs (TRNPs). When mouse oocytes were treated with NPs, only TRNPs could fully pass through the ZP and cell membrane. To assess the effects of TRNPs on fertility and potential nanotoxicity, we performed mRNA sequencing analysis to confirm their genetic safety. We established a system to successfully internalize TRNPs into oocytes. The genetic stability and normal development of TRNP-treated oocytes and embryos were confirmed. These results imply that TRNPs can be used as a drug delivery carrier applicable to germ cells.
Influence of Lysine and TRITC Conjugation on the Size and Structure of Dextran Nanoconjugates with Potential for Biomolecule Delivery to Neurons
ACS Appl Bio Mater2021 Sep 20;4(9):6832-6842.PMID: 35006983DOI: 10.1021/acsabm.1c00544
As a potent nonviral system for biomolecular delivery to neurons via their axons, we have studied molecular characteristics of lysinated fluorescent dextran nanoconjugates with degrees of conjugation of 0.54-15.2 mol lysine and 0.25-7.27 mol tetramethyl rhodamine isothiocyanate (TRITC) per mol dextran. We studied the influence of conjugation with lysine and TRITC on the size and structure of different molecular weight dextrans and their mobility within axons. Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) experiments revealed significant differences in the size and structure of unmodified and modified dextrans. Unexpectedly, lower-molecular-weight conjugated dextrans exhibited higher molecular volumes, which we propose is due to fewer intramolecular interactions than in higher-molecular-weight conjugated dextrans. Assessment of retrograde and anterograde movement of lysine- and TRITC-conjugated dextrans in axons in the lumbar spinal cord of chicken embryos showed that lower-molecular-weight dextrans translocate more efficiently than higher-molecular-weight dextrans, despite having larger molecular volumes. This comparative characterization of different molecular weight dextrans will help define optimal features for intracellular delivery.
Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5⁺ stem cell
Nat Med2012 Mar 11;18(4):618-23.PMID: 22406745DOI: 10.1038/nm.2695
Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5(+) cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP(+) colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5(+) colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.
Hollow carbon dots labeled with FITC or TRITC for use in fluorescent cellular imaging
Mikrochim Acta2018 Mar 14;185(4):223.PMID: 29594848DOI: 10.1007/s00604-018-2761-2
Hollow carbon dots (HCDs) were prepared by a solvothermal method and conjugated to either tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein-5-isothiocyanate (FITC). This resulted in HCDs with bright red or green fluorescence, with excitation/emission peaks at 550/580 and 491/520 nm, respectively. The nanocomposites are well water-soluble, remarkably photostable and biocompatible. In addition, the fluorescence of the composites is more stable in a reactive oxygen environment than the free dyes. Confocal images indicate that the nanoparticles quickly enter A549 cells and mainly accumulate in the cytoplasm. The wavelength of functionalized HCDs can be regulating via coupling the HCDs to different dyes. These results demonstrate that these composite materials can be very promising reagents for biological labeling and imaging. Graphical abstract Schematic of the preparation of hollow carbon dots conjugated to tetramethyl rhodamine isothiocyanate (RHCDs) by solvothermal method. The material is water-soluble, remarkably photostable and biocompatible. It was applied to cellular labeling and imaging.