Phalloidin-The principle and experimental procedure of cell skeleton staining
The cell skeleton is a reticular fiber structure that is highly dynamic and exhibits different forms at different stages. It is mainly composed of microfilaments, microtubules, and intermediate filaments. Microfilaments are primarily composed of actin, which exists in two forms: globular actin (G-actin) and filamentous actin (F-actin). The assembly of microfilaments involves the transformation of G-actin into F-actin.
Figure 1. Cell skeleton structure (including microfilaments, microtubules, and intermediate filaments). Image source: "Medical Cell Biology: Chapter 7 Cell Skeleton and Cell Movement"
The phalloidin is a cyclic heptapeptide, one of the earliest cyclic peptides discovered, isolated from the Amanita phalloides. It functions by binding to and stabilizing filamentous actin (F-actin) effectively preventing the depolymerization of actin filaments. Due to its tight and selective binding to F-actin, it reveals the distribution of the microfilament skeleton within cells, thus fluorescence labeling with phalloidin has been widely used in biological microscopy techniques.
Figure 2. Structure of Phalloidin.
It is worth noting that unmodified phalloidin cannot penetrate the cell membrane, making it difficult to directly apply in experiments involving live cells. Phalloidin labeled with fluorescent substances such as FITC and Rhodamine can specifically bind to F-actin in eukaryotic cells, thus revealing the distribution of the microfilament skeleton within cells.
Arcegen Biosciences offers a wide variety of phalloidin products, primarily distinguished by different fluorescent tags, resulting in different fluorescence colors upon excitation. This diversity caters to the varying requirements of excitation and emission wavelengths by different machines, allowing for differentiation of fluorescence colors when co-staining with other fluorescent dyes.
Experimental Procedure
The preparation of phalloidin
0.1 mg of phalloidin dissolved in 1 ml of anhydrous methanol (or DMSO or anhydrous ethanol) to prepare a stock solution. The stock solution can be stored long-term at -20°C in the dark. The working concentration is 5 μg/ml, which can be achieved by diluting the stock solution 20-fold with PBS before use.
Staining Procedure
PBS wash cells twice for 10 minutes each time; fix cells with 3.7%-4% formaldehyde or paraformaldehyde for 20 minutes, followed by PBS washing cells twice; stain cells with 5 μg/ml FITC-phalloidin at room temperature for 30-60 minutes (shorter duration in summer), followed by PBS washing cells twice; stain cell nuclei with DAPI or Hoechst 33258 for 10 minutes, followed by PBS washing cells twice; remove excess moisture, add fluorescence mounting medium (neutral or slightly alkaline buffer mixed with an equal amount of glycerol), cover with a cover slip, and observe under a fluorescence or confocal microscope.
Customer examples
Customer Source: Sichuan University West China Hospital Dental Clinic
Cell Type: Bone Marrow Mesenchymal Stem Cells
Scale: 100 μm
Reference: Zhu Z, Jiang S, Liu Y, et al. Micro or nano: Evaluation of biosafety and biopotency of magnesium metal organic framework-74 with different particle sizes. Nano Research, 2020, 13(2): 511-526.
Product Introduction: The staining product used is FITC-labeled phalloidin, which stains cells with green fluorescence. The image shows the determination of the safe dose of Mg-MOF74 for BMSCs using the MTT assay, FITC-labeled phalloidin staining, and DAPI staining, ensuring subsequent experiments are conducted within a safe dose range.
Cell Type: Bone Marrow Mesenchymal Stem Cells
Scale: 50 μm
Reference: Fan Z, Xiao L, Lu G, Ding Z, Lu Q. Water-insoluble amorphous silk fibroin scaffolds from aqueous solutions. J Biomed Mater Res B Appl Biomater. 2020;108(3):798-808. doi:10.1002/jbm.b.34434
Product Introduction: The staining product used is rhodamine-labeled phalloidin, which stains cells with red fluorescence. The image shows rhodamine-labeled phalloidin staining and DAPI nuclear staining, observing the differentiation of BMSCs into endothelial cells on different regenerated silk fibroin scaffolds under confocal microscopy.
Summary: Fluorescent and biotinylated phalloidin derivatives have significant advantages over actin antibodies. The binding of phalloidin conjugates remains unchanged with variations in actin across species (including animals and plants). Moreover, their nonspecific staining is negligible. Therefore, the contrast between stained and unstained areas is high.
Precautions
Phalloidin has a small molecular weight and can easily penetrate cells, so there is no need for cold acetone or TRITON X-100 treatment of cells. It is highly toxic, so wear gloves when handling.
Product features
High affinity: Kd= 20 nM;
Strong specificity: selectively binds to filamentous actin (F-actin) without binding to monomeric actin (G-actin);
Superior to antibody staining: Phalloidin has no species limitations and almost no nonspecific staining, resulting in extremely clear contrast between stained and unstained areas;
High sensitivity: staining at nanomolar concentrations (nM) is sufficient for experimental requirements;
Good compatibility with actin activity unaffected: Phalloidin derivatives are small molecules, approximately 12-15Å in diameter, with a molecular weight <2000 Da, allowing many physiological properties of labeled actin to be maintained;
Wide applicability: No species differences; equally suitable for formaldehyde-fixed and permeabilized tissue samples.
FAQ
Q1: Can phalloidin staining be performed in live cells?
A: Phalloidin staining requires sample fixation and permeabilization to facilitate binding between phalloidin and F-actin, resulting in better staining results.
Q2: My cell samples are transfected with a plasmid containing GFP. Which type of phalloidin should I choose?
A: You can choose phalloidin labeled with TRITC (orange), iFluorTM 555 (orange), or iFluorTM 647 (far-red).
Q3: For adherent cells, how much working solution is needed per sample?
A: Adherent cell staining only requires enough working solution to completely cover the cells.
Q4: What are the differences between different types of phalloidin, and how should I choose?
A: The differences lie in the fluorescent tags, resulting in different fluorescence colors. This aids in distinguishing fluorescence colors of other co-stained fluorescent dyes. When choosing, ensure the machine meets the excitation and emission wavelength requirements.
Q5: Is there species specificity in phalloidin staining?
A: There is no species specificity for staining live animal cells.
Related Products
|
Product Name |
Catalog Number |
Specification |
|
Phalloidin |
C331201E |
1 mg |
|
Phalloidin |
C331201S |
5 mg |
|
Amino-Phalloidin |
C331202E |
1 mg |
|
TRITC Phalloidin |
C331207E |
300 T |
|
TRITC Phalloidin |
C331207S |
1 mg |
|
FITC Phalloidin |
C331203E |
300 T |
|
FITC Phalloidin |
C331203S |
1 mg |
|
Fluor 488 phalloidin, Green |
C331204E |
300 T |
|
Fluor 555 phalloidin, Orange |
C331205E |
300 T |
|
Fluor 647 phalloidin, Far Infrared |
C331206E |
300 T |