FITC Phalloidin FITC标記鬼筆環肽

産品标簽

Phalloidin 鬼筆環肽；F-actin 肌動蛋白(bái)（聚合）；G-actin 球形肌動蛋白(bái)（單體）；Actin Polymerization 肌動蛋白(bái)聚合；Cytochalasin 細胞松弛素；CAS NO.：17466-45-4；

産品信息

【溫馨提示】：見(jiàn)我司整理的鬼筆環肽及衍生(shēng)物(wù)（Phalloidin and Phalloidin Conjugates）産品專題。

産品描述

鬼筆環肽（Phalloidin），又(yòu)稱鬼筆鵝膏素，最初是從(cóng)毒蘑菇鬼筆鵝膏（Amanita phalloides）中分離到(dào)的一(yī)種七肽毒素，以極高(gāo)的親和力和特異性結合肌動蛋白(bái)絲F-actin（聚合形式的肌動蛋白(bái)），不會(huì)結合單體肌動蛋白(bái)（G-actin）。不像肌動蛋白(bái)抗體，同時識别單體和聚合形式的肌動蛋白(bái)。鬼筆環肽對大小(xiǎo)纖維的親和力相(xiàng)近，在許多(duō)不同的動植物(wù)物(wù)種的肌肉和非肌肉細胞中，基本都按照(zhào)一(yī)個(gè)肌動蛋白(bái)亞基與一(yī)個(gè)鬼筆環肽分子的化學計量比結合。不像肌動蛋白(bái)抗體，對不同物(wù)種或來源的肌動蛋白(bái)親和力會(huì)發生(shēng)明顯變化。鬼筆環肽的非特異性結合幾乎可忽略，染色和未染色區域的差異極其明顯。鬼筆環肽使得肌動蛋白(bái)聚合/解離的臨界濃度降至1μg/ml，可用作一(yī)種聚合增強劑。另外，産生(shēng)的複合物(wù)高(gāo)度穩定（解離常數約3×10–8M），能(néng)夠抑制細胞松弛素、碘化鉀和溫度上(shàng)升引起的去聚合和去組裝活性。

鬼筆環肽及其衍生(shēng)物(wù)在納摩爾濃度即可對F-actin染色，且水(shuǐ)溶性良好，是非常實用和方便的探針，對組織切片、細胞培養物(wù)或無細胞體系内的F-actin進行定性和定量研究。另外，鬼筆環肽及其衍生(shēng)物(wù)很小(xiǎo)，直徑約12–15 Å，分子量<2000 Da，經标記後的F-actin仍維持許多(duō)标記前的功能(néng)。比如，标記的甘油抽提肌纖維仍能(néng)收縮；标記的肌動蛋白(bái)絲仍能(néng)在固相(xiàng)肌球蛋白(bái)基質中移動。

本品為(wèi)FITC熒光(guāng)标記的鬼筆環肽（FITC-Phalloidin），以溶于甲醇的20µM儲存液形式提供，具體的工(gōng)作濃度請參考文獻或自(zì)身實驗體系來調整，常用濃度範圍80-200nM。按照(zhào)100nM的工(gōng)作濃度來算(suàn)，可進行300次細胞染色。

産品應用

1）對固定的組織切片和組織細胞培養物(wù)進行肌動蛋白(bái)絲（F-actin）的熒光(guāng)染色；

2）體外制備穩定的熒光(guāng)肌動蛋白(bái)絲（F-actin）；

保存與運輸方法

保存：-20℃避光(guāng)保存，1年(nián)有效。

運輸：冰袋運輸。

注意事(shì)項

1）本品具有一(yī)定的毒性，LD50=2mg/kg，操作時請注意防護。

2）本品（Cat#MX4403）以溶于甲醇的儲存液（20µM）形式提供，300T包裝，适合用量少的研究者；另提供凍幹粉形式（Cat#MX4404），1mg包裝，相(xiàng)對更經濟，适合用量多(duō)的研究者；

3）為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

操作流程（免疫熒光(guāng)染色）

有幾種方法都能(néng)用來染色組織細胞培養物(wù)内的肌動蛋白(bái)絲（F-actin）染色，其中，固定步驟在獲得可信且具代表性的細胞F-actin分布情況中至關重要。固定步驟基于實驗自(zì)身需求來選擇。多(duō)聚甲醛或戊二醛都能(néng)得到(dào)優秀的F-actin染色和良好的闆狀僞足維持保存。

一(yī)、實驗材料準備

1.1 FITC Phalloidin（Cat# MX4403）

1.2 1×PBS Buffer（pH 7.4）, for Cell Culture（Cat#SH30256.01）

1.3 固定液（溶于PBS的4%多(duō)聚甲醛，pH 7.0）（Cat# MM1504）

1.4 破膜液（溶于PBS的0.5% Triton X-100）

1.5 抗熒光(guāng)淬滅劑（Cat# MM1401或Cat# MM1402）

1.6 即用型DAPI染色液（Cat# MX4209）

1.8 （可選）BSA, Standard Grade（Cat# MP6101）

1.9 蓋玻片密封液（透明指甲油）

二、染色工(gōng)作液準備

2.1 剛收到(dào)或第一(yī)次使用時，将充分融化的本品（20µM儲存液）根據單次使用量進行分裝，置于-20℃避光(guāng)保存，一(yī)年(nián)穩定。

2.2 正式實驗前，使用1×PBS Buffer（pH 7.4）稀釋儲存液到(dào)需要的工(gōng)作濃度，常用的工(gōng)作濃度範圍為(wèi)80-200nM。可以100nM為(wèi)起始工(gōng)作濃度，最佳的工(gōng)作濃度請參考文獻或根據實驗室現有體系來摸索。

【注①】：可使用含1% BSA的PBS Buffer稀釋儲存液，能(néng)夠降低(dī)非特異背景染色，也能(néng)最小(xiǎo)化鬼筆環肽粘附到(dào)管壁的可能(néng)性。

【注②】：染色工(gōng)作液現配現用，室溫避光(guāng)保存。

三、染色流程

3.1 細胞爬片培養，使其密度至少達半彙合。

3.2 吸掉培養液，用37℃預熱的1×PBS Buffer（pH 7.4）清洗細胞2次。

3.3 用溶于PBS的4%多(duō)聚甲醛固定細胞，室溫固定10min。【注意】：固定過程中甲醇能(néng)破壞肌動蛋白(bái)，因此，固定液中最好避免接觸任何甲醇。最好的固定液是無甲醇的甲醛。

3.4 用1×PBS Buffer清洗細胞2-3次，室溫下(xià)30s/次。

3.5 用破膜緩沖液透化細胞，室溫處理5min。

3.6 用1×PBS Buffer清洗細胞2-3次，室溫下(xià)30s/次。

3.7 取200μl現配的FITC-Phallodin染色工(gōng)作液，完全覆蓋蓋玻片上(shàng)的細胞，室溫避光(guāng)孵育30min。【注意】：通(tōng)常情況4℃-37℃都适合用于染色。為(wèi)了避免工(gōng)作液的揮發，孵育過程中将蓋玻片置于一(yī)密封的容器(qì)内。

3.8 用1×PBS Buffer清洗細胞2-3次，室溫下(xià)30s/次。

3.9 取200μl DAPI溶液（100nM）或商業(yè)化的即用型DAPI染色液對細胞核複染，室溫約30s。

3.10 用1×PBS Buffer清洗細胞1-2次，室溫下(xià)30s/次。

3.11 将蓋玻片倒置在滴有一(yī)滴抗熒光(guāng)淬滅劑（如Fluoromount-GTM，Cat# MM1401）的載玻片上(shàng)。用紙(zhǐ)巾輕輕吸掉多(duō)餘淬滅劑，然後用透明指甲油密封蓋玻片四周。将此法處理玻片置于4℃避光(guāng)存放(fàng)至少6個(gè)月(yuè)仍能(néng)維持F-actin染色。

【可選】：完成步驟3.8後，直接将蓋玻片倒置在含DAPI的抗熒光(guāng)淬滅劑（如DAPI Fluoromount-GTM，Cat# MM1402）的載玻片上(shàng)。然後吸掉多(duō)餘淬滅劑并用指甲油密封蓋玻片四周。

3.12 熒光(guāng)顯微鏡下(xià)觀察染色結果，選擇FITC激發/發射濾片（Ex/Em=492/518nm）和DAPI激發/發射濾片（Ex/Em=364/454nm）。

相(xiàng)關産品

 — —Written/Edited by V. Shallan【版權歸MKBio懋康所有】

上海夢澤生物科技有限公司是一(yī)家涉足于生(shēng)命科學和生(shēng)物(wù)技(jì)術(shù)領域研究的試劑、儀器(qì)和實驗室消耗品與實驗服務工(gōng)作，主要從(cóng)事(shì)細胞生(shēng)物(wù)學、植物(wù)學、分子生(shēng)物(wù)學、免疫學、生(shēng)物(wù)化學、蛋白(bái)組學。生(shēng)物(wù)制藥與診斷試劑研發生(shēng)産等領域。 本公司秉承“以人為(wèi)本，以誠為(wèi)信、合同守信”的經營理念。堅持"品質保障"的原則為(wèi)廣大客戶提供優質産品。

文獻引用：

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Qian Xu et al. Novel injectable and self-setting composite materials for bone defect repair. SCIENCE CHINA Materials, Volume 63, Issue 5: 876-887(2020)

MC3T3-E1 cells were first inoculated into six-well plates at the initial density of 4×104 cells/well (4 mL per well) for 24 h. The cell medium was replaced by 4 mL of the extraction medium when the cell confluence reached approximately 80%. Cells were gently washed by PBS and fixed with 4% polyformaldehyde for 20 min, followed by PBS washing for three times. A fluorescein phalloidin (FITC-phalloidin) solution (10 µg mL−1) (Mao Kang Biotechnology Co., Ltd., Shanghai, China) was added to the wells and stained for 50 min. The samples were subsequently washed with PBS three times and stained with a 4ʹ,6-diamidino-2-phenylindole (DAPI) solution (100 ng mL−1) to counterstain the cell nuclei for 15 min. These samples were finally washed with PBS before observation under an Eclipse Ti inverted fluorescence microscope (Nikon Instruments Inc., Tokyo, Japan).

（2）

Han Y, Lian M, Sun B, Jia B, Wu Q, Qiao Z, Dai K. Preparation of high precision multilayer scaffolds based on Melt Electro-Writing to repair cartilage injury. Theranostics 2020; 10(22):10214-10230. doi:10.7150/thno.47909.

After 21 days of culturing, the cell-bearing scaffolds (n = 4 per group) were fixed in 4% paraformaldehyde for 2 h. Triton-X (0.1%) was used to soak the scaffolds for 5 min, which were then washed thrice with PBS for 5 min each. Blocking was performed with BSA at room temperature for 1 h. After blotting with water-absorbing paper, primary antibodies (anti-PRG4, anti-CILP, anti-COLII, anti-COLI, and anti-SOX-9) (1:100, Abcam, UK) were added to each sample and incubated at 4 °C overnight. Following washes with PBS, a corresponding secondary antibody (1: 500, Abcam, UK) was added, incubated at room temperature for 1 h, and washed with PBS. The scaffolds were then labeled with FITC phalloidin (1:200, MaoKang, China) and incubated at room temperature for 2 h, followed by incubation with DAPI staining solution (1:1000, MaoKang, China) for 30 min at room temperature. After the final wash with PBS, samples were observed and imaged under a confocal microscope (LEICA, Germany). Analysis and quantification of protein expression was performed using ImageJ.

(3)

Li Chengpan et al. On-Chip Replication of Extremely Early-Stage Tumor Behavior. ACS Appl. Mater. Interfaces 2021, 13, 17, 19768–19777

Phalloidin-FITC, and DAPI dyes were purchased from Shanghai Maokang Biotechnology Ltd.

[4]
Ming, Ping Deng, et al. “Preparation and Characterization of Silk Fibroin-Based Hybrid Vascular Tissue Engineering Film.” Materials Science Forum, vol. 996, Trans Tech Publications, Ltd., June 2020, pp. 64–69. Crossref, doi:10.4028/www.scientific.net/msf.996.64.

FITC Phalloidin was purchased from Shanghai Mao Kang biotechnology Co., Ltd., ( Shanghai, China) 

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Cheng panLi et al. Alginate core–shell microcapsule reduces the DMSO addition-induced osmotic damage to cells by inhibiting cellular blebs. Chinese Journal of Chemical Engineering. Volume 33, May 2021, Pages 249-255

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Each sample was stained with DAPI and FITC-phalloidin reagents (Shanghai Mao Kang Biotechnology Co. Ltd).

[6] Wei Cui, Qibin Song, Huhu Su, Zhiqing Yang, Rui Yang, Na Li, Xing Zhang. Synergistic effects of Mg-substitution and particle size of chicken eggshells on hydrothermal synthesis of biphasic calcium phosphate nanocrystals[J]. Journal of Materials Science & Technology, 2020, 36(0): 27-36

https://doi.org/10.1016/j.jmst.2019.04.038

MC3T3-E1 cells with the initial density of 10⁴ cells/cm² were placed in culture plates. After culture with BCP extracts using different particle sizes of 45 μm, 45-75 μm, 150-840 μm and without BCP extracts (control group) for 24 h, cells washed with phosphate buffered saline (PBS) were fixed with 4% paraformaldehyde at 4 °C for 15 min. The cells were stained with 10 ug/ml FITC-phalloidin (Mao Kang, Biotechnology, co, LTD, Shanghai, China) for 50 min and counterstained with 100 mg/ml 4′,6-diamidino-2-phenylindole (DAPI, Dingguo Changsheng Biotechnology, co, LTD, Beijing, China) for 15 min at room temperature after washed with PBS for three times. Finally, the fluorescent staining of cells was observed by an Eclipse Ti fluorescence inverted microscope (Nikon Instruments Inc., Tokyo, Japan).

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LeiCao et al. Plasma spray of biofunctional (Mg, Sr)-substituted hydroxyapatite coatings for titanium alloy implants. Journal of Materials Science & Technology. Volume 35, Issue 5, May 2019, Pages 719-726

MC3T3-E1 cells were placed in culture plates at an initial density of 1 × 10⁴ cells/cm². After culture for 48 h, cells were washed with phosphate buffered saline (PBS) three times, and fixed with 4% paraformaldehyde at 4 °C for 15 min. The fixed samples were then washed with PBS three times and stained with 10 μg/ml FITC-phalloidin (Mao Kang, Biotechnology, co, LTD, Shanghai, China) for 50 min at room temperature.

  Fig. 7. Immunofluorescence staining of MC3T3-E1 cells cultured with (a, b) normal culture medium and (c, d) (Mg, Sr)-HA coating extract for 48 h. (b, d) show the partially enlarged images from the white dash boxes from (a, c). The cell nuclei and smooth muscle alpha-actin are stained with DAPI (blue) and FITC-phalloidin (green), respectively.

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Huo-Liang Zheng, Wen-Ning Xu, Peng-Bo Chen, Lei-Sheng Jiang, Xin-Feng Zheng, Sheng-Dan Jiang, "Increased Expression of Prolyl Endopeptidase Induced by Oxidative Stress in Nucleus Pulposus Cells Aggravates Intervertebral Disc Degeneration", Oxidative Medicine and Cellular Longevity, vol. 2022, Article ID 9731800, 16 pages, 2022. https://doi.org/10.1155/2022/9731800

 DAPI (Beyotime) and FITC-conjugated phalloidin (MKBio) were used to stain the nuclei and cytoskeletons. Images were obtained using an Olympus microscope.