TRITC Phalloidin

Background

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]

参考文献:
[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]

Protocol

1.?? Prepare 1× TRITC Phalloidin working solution:

Add 1 μL of 1000× TRITC Phalloidin DMSO solution to 1 mL of PBS containing with 1% BSA.

Note: 1. Different cell types might be stained differently. The concentration of TRITC Phalloidin working solution should be prepared accordingly.

???? 2. ?1% bovine serum albumin (BSA) is used to reduce nonspecific background staining.

?

2.?? Stain the cells:

2.1?? Wash cells 2–3 times with PBS. Fix the cells in 3.7% methanol-free formaldehyde solution in PBS for 10-30 minutes at room temperature.

Note: Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.

2.2?? Wash the fixed cells 2–3 times in PBS.

2.3?? Optional: Add 0.1% Triton X-100 in PBS into fixed cells for 3–5 minutes to increase permeability. Wash the cells 2–3 times in PBS.

2.4?? Add 100 μL/well (96-well plate) of TRITC Phalloidin working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.

2.5?? Rinse cells gently with PBS 2–3 times to remove excess TRITC Phalloidin.

2.6?? Run fluorescence microscope or other equipments at desired Ex/Em wavelengths (TRITC Phalloidin, Ex/Em=552/578 nm).