FITC Phalloidin FITC  標記鬼筆環肽   （金標產品，享發文獎勵）

產品標簽

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

產品信息

【溫馨提示1】：見我司整理的鬼筆環肽及衍生物（Phalloidin and Phalloidin Conjugates）產品專題。

【溫馨提示2】：自2024年9月開始FITC-phallodin更換成DMSO儲存液（1000×）形式提供，按照100μl/孔（96孔板），總共可做300次。

產品描述

鬼筆環肽（Phalloidin），又稱鬼筆鵝膏素，最初是從毒蘑菇鬼筆鵝膏（Amanita phalloides）中分離到的一種七肽毒素，以極高的親和力和特異性結合肌動蛋白絲F-actin（聚合形式的肌動蛋白），不會結合單體肌動蛋白（G-actin）。不像肌動蛋白抗體，同時識別單體和聚合形式的肌動蛋白。

鬼筆環肽對大小纖維的親和力相近，在許多不同的動植物物種的肌肉和非肌肉細胞中，基本都按照一個肌動蛋白亞基與一個鬼筆環肽分子的化學計量比結合。不像肌動蛋白抗體，對不同物種或來源的肌動蛋白親和力會發生明顯變化。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聚合/解離的臨界濃度降至1μg/ml，可用作一種聚合增強劑。另外，產生的復合物高度穩定（解離常數約3×10–8M），能夠抑制細胞松弛素、碘化鉀和溫度上升引起的去聚合和去組裝活性。

鬼筆環肽及其衍生物在納摩爾濃度即可對F-actin染色，且水溶性良好，是非常實用和方便的探針，對組織切片、細胞培養物或無細胞體系內的F-actin進行定性和定量研究。另外，鬼筆環肽及其衍生物很小，直徑約12–15 ?，分子量<2000 Da，經標記后的F-actin仍維持許多標記前的功能。比如，標記的甘油抽提肌纖維仍能收縮；標記的肌動蛋白絲仍能在固相肌球蛋白基質中移動。

本品為FITC熒光標記的鬼筆環肽（FITC-Phalloidin），以溶于DMSO的儲存液形式提供，按照100μl/孔（96孔板），總共可做300次。

產品應用

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

2） 體外制備穩定的熒光肌動蛋白絲（F-actin）；

保存與運輸方法

保存：-20℃避光保存，1年有效。 

運輸：冰袋運輸。

注意事項

1） 本品具有一定的毒性，LD50=2mg/kg，操作時請注意防護。

2） 為了您的安全和健康，請穿實驗服并戴一次性手套操作。

操作流程（免疫熒光染色）

有幾種方法都能用來染色組織細胞培養物內的肌動蛋白絲（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身需求來選擇。多聚甲醛或戊二醛都能得到優秀的F-actin染色和良好的板狀偽足維持保存。

一、 實驗材料準備

1.1 FITC Phalloidin（Cat# MX4403）

1.2 1×PBS Buffer（pH 7.4）, for Cell Culture（Cat# MS3552）

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

1.4 破膜液（溶于PBS的0.5% Triton X-100）

1.5 抗熒光淬滅劑（Cat# MM1401或Cat# MM1402）

1.6 即用型DAPI染色液（Cat# MX4209）

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

1.8 蓋玻片密封液（透明指甲油）

二、 染色工作液準備

2.1于實驗前，將低溫保存的FITC-Phalloidin置于室溫回溫至少20min，低速離心后才開瓶。第一次使用請將本品按照單次用量進行分裝，置于-20℃密封避光干燥保存，一年穩定。

2.2本品以1000×DMSO儲存液形式提供。開始實驗前，使用含1% BSA的1×PBS Buffer（pH 7.4）將其稀釋到1×染色工作液（比如：1μl儲存液加入1ml含1% BSA的PBS Buffer），用槍吹勻即可。

【注①】：以96孔板為檢測體系，按照100μl/孔來計算，準備足量的染色工作液；也可對細胞爬片進行染色，但需調整染色工作液的用量，以能覆蓋住細胞為準。

【注②】：最佳的工作濃度和孵育時間取決于特定的應用（初次實驗，可在1:1000~1:2000的稀釋倍數范圍內進行濃度優化）。根據特定的細胞類型和/或細胞/組織對探針的通透性來調整合適的染色條件。

【注③】：建議使用含1% BSA的PBS Buffer稀釋儲存液，能夠降低非特異背景染色，也能最小化鬼筆環肽粘附到管壁的可能性。若無現成的BSA，可替代使用1×PBS Buffer（pH 7.4），以能得到理想結果為宜。

【注④】：染色工作液現配現用，室溫避光保存。

三、 染色流程（以96孔板為例）

3.1用黑框/透明底的96孔板進行細胞培養，使其貼壁生長達到70-80%的匯合度。

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%多聚甲醛固定細胞，室溫固定10-30min?！咀⒁狻浚汗潭ㄟ^程中甲醇能破壞肌動蛋白，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30s/次。

3.5【可選】用破膜緩沖液透化細胞以提高染料的滲透性，室溫處理5min。

3.6用1×PBS Buffer清洗細胞2-3次，室溫下30s/次。

3.7 取100μl現配的FITC-Phallodin染色工作液到每個孔內，完全覆蓋細胞，室溫避光孵育20-90min。

【注意】：通常情況4℃-37℃都適合用于染色。

3.8用1×PBS Buffer清洗細胞2-3次，室溫下30s/次。

3.9【可選】加入100μl 即用型DAPI染色液或其他不同于FITC熒光光譜的細胞核染色液對細胞核復染，室溫約5min?！咀⒁狻浚杭毎藦腿緯r間可自行調整；細胞核復染也可以放到步驟3.7中進行。

3.10用1×PBS Buffer清洗細胞2-3次，室溫下30s/次。

3.11【可選】加抗熒光淬滅劑（如Fluoromount-GTM，Cat# MM1401）到孔內保護熒光。

3.12 熒光顯微鏡下觀察染色結果，選擇FITC激發/發射濾片（Ex/Em=492/518nm）。若做細胞核染色，選擇合適濾片，比如DAPI激發/發射濾片（Ex/Em=364/454nm）。

相關產品

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

上海懋康生物科技有限公司是一家涉足于生命科學和生物技術領域研究的試劑、儀器和實驗室消耗品與實驗服務工作，主要從事細胞生物學、植物學、分子生物學、免疫學、生物化學、蛋白組學。生物制藥與診斷試劑研發生產等領域。 本公司秉承“以人為本，以誠為信、合同守信”的經營理念。堅持"品質保障"的原則為廣大客戶提供優質產品。

文獻引用：

（1）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 (4mL per well) for 24h. The cell medium was replaced by 4mL of the extraction medium when the cell confluence reached approximately 80%. 

Cells were gently washed by PBS and fixed with 4% polyformaldehyde for 20min, followed by PBS washing for three times.A fluorescein phalloidin (FITC-phalloidin) solution (10μgmL?1) (Mao Kang Biotechnology Co., Ltd., Shanghai, China)was added to the wells and stained for 50min. The samples were subsequently washed with PBS three times and stained with a 4?,6-diamidino-2-phenylindole (DAPI) solution (100ngmL?1) to counterstain the cell nuclei for 15min. 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) 

[5]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

[6] Xia, C., Ming, P., Zhou, A. et al. Supramolecular self-assembly of oligopeptide hybrid films with liquid crystal texture: effects on cell behaviour for vascular grafts. Bull Mater Sci 44, 197 (2021).https://doi.org/10.1007/s12034-021-02470-x

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

[7] 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

[8] 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).

[9]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 andstained 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.

[10]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.