One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit is a ready-to-use kit that enables specific dual labeling of F-actin and cell nuclei simultaneously. Its core components are LumiDye™ Phalloidin and Nucleiblue, engineered to meet experimental requirements for co-localization analysis of the cytoskeleton and cell nuclei. Core Working Principles:
1. As a phalloidin derivative, LumiDye™ Phalloidin binds specifically and with high affinity to filamentous actin (F-actin) in eukaryotic cells, with no binding to monomeric G-actin. It precisely labels the microfilament structures of the cytoskeleton.
2. Nucleiblue is a highly specific nuclear fluorescent dye that selectively binds to nuclear DNA for distinct fluorescent labeling of cell nuclei. Combined application of the two reagents achieves synchronous staining of F-actin and nuclei.
Unlike conventional separately prepared actin and nuclear staining reagents, this kit adopts a ready-to-use pre-mixed system. Researchers are spared the steps of self-dilution and formulation, drastically cutting experimental operation time. The optimized reagent ratio minimizes non-specific staining to negligible levels. In addition, LumiDye™ Phalloidin features a low molecular weight; its binding to F-actin does not interfere with interactions between actin-binding proteins, thereby preserving the biological functions of F-actin. LumiDye™ dyes represent a new generation series independently developed by our company. Compared with alternative fluorescent dyes, they deliver comprehensive superiorities in brightness, photostability and water solubility. Specifically, LumiDye™ Phalloidin stabilizes F-actin structures and prevents depolymerization to fully preserve intact microfilament morphology. It exhibits no notable disparity in binding affinity toward F-actin across different species and barely disrupts F-actin functionality. Labeled actin filaments retain normal physiological functions: stained muscle fibers remain contractile, and tagged myosin maintains motility. It binds actin subunits at a 1:1 stoichiometric ratio with comparable affinity for thick and thin microfilaments in diverse plant and animal cells, supporting direct quantitative analysis of cellular F-actin. Nucleiblue produces intensely bright fluorescent signals with robust photostability and ultra-low background interference, enabling clear visualization of nuclear morphology, location and numerical variation. These multifold advantages facilitate efficient qualitative and quantitative analysis. This kit is widely compatible with observation under fluorescence microscopes (including laser scanning confocal microscopes) and flow cytometry detection. It suits experimental scenarios such as fundamental cell biology research and cytoskeleton dynamics analysis.
As a pivotal component of the cytoskeleton, filamentous F-actin’s morphology and distribution are tightly correlated with structural alterations of cell nuclei, serving as critical biomarkers for cell migration and morphological remodeling. Phalloidin has long been a gold-standard tool for actin staining due to its specific F-actin binding capacity, while Nucleiblue is a novel high-specificity nuclear fluorescent stain and a top-choice reagent for nuclear labeling. Co-staining with the two constitutes a core analytical method for cellular morphology and subcellular structure assessment.
Against this scientific backdrop, our kit integrates optimized formulations of LumiDye™ Phalloidin and Nucleiblue. LumiDye™ Phalloidin combines precise labeling, intact functional retention and compatibility with quantification. Under fluorescence microscopy, it clearly visualizes the spatial relationship between F-actin microfilament distribution and nuclei for accurate subcellular co-localization analysis. In flow cytometry, it allows simultaneous precise quantification of F-actin expression levels. For drug screening studies, it rapidly detects cytoskeleton remodeling and nuclear morphological shifts post-drug treatment, providing intuitive morphological evidence for investigating drug mechanisms of action. In basic cell biology research, it characterizes dynamic changes in F-actin and nuclei during cell migration, invasion and differentiation, allowing investigations while preserving F-actin physiological functions to maximally restore the native cellular physiological state. Boasting core strengths of high specificity, exceptional stability, superior user-friendliness and full functional preservation, this product provides researchers with an efficient, reliable dual-labeling tool for cytoskeleton-nucleus analysis.
Application Scope:
Labeling of F-actin in cells, frozen tissue sections, yeast and fungi; compatible with immunofluorescence staining.
Product Features:
⁕ Integrated dual labeling, ready-to-use formulation
Premixed staining reagents of LumiDye™ Phalloidin and Nucleiblue for direct use upon opening. No dilution, blending or titration required. One-step staining for both F-actin and cell nuclei, greatly simplifying experimental procedures.
⁕ Precise signals, accurate co-localization
LumiDye™ Phalloidin binds F-actin with high specificity and does not recognize G-actin; Nucleiblue labels cell nuclei accurately with bright signals and low background.
⁕ Broad applicability, beginner-friendly
Time-saving and labor-efficient with reduced experimental steps and reagent consumption. Easy operation for new users. Compatible with coverslip cultures, routine microscopic observation, batch samples, 96-well plates, high-content screening and automated experimental platforms, suitable for high-throughput assays and all routine research experiments.
⁕ Species-independent binding
Unlike antibodies, phalloidin exhibits no significant difference in binding affinity to F-actin across various species, with negligible non-specific staining.
⁕ Function preservation, quantitation compatible
Hardly interferes with the physiological functions of F-actin. Labeled muscle fibers remain contractile and myosin maintains normal motility. It binds actin subunits at a 1:1 stoichiometric ratio with equal affinity for microfilaments in all cell types, supporting quantitative analysis of F-actin.
⁕ Stable results, accurate imaging
Fixed dye ratio and uniform reaction system minimize batch-to-batch and operator-induced variations, delivering excellent staining reproducibility. Simultaneous staining avoids cell displacement and elution issues caused by sequential incubation, yielding more reliable co-localization data, superior signal-to-noise ratio and uniform, steady imaging performance.
Product Specifications:
Item No.
| Product Name
| 50T | 300T | LumiDye™ Ex/Em | Nucleiblue Ex/Em | | O1524682 | One-Step LumiDye™ 488 Phalloidin/Nucleiblue Double Staining Kit
| 5 mL | 30 mL | 490/515 nm | 345-355/460-470 nm | | O1524683 | One-Step LumiDye™ 555 Phalloidin/Nucleiblue Double Staining Kit
| 5 mL | 30 mL | 555/565 nm | 345-355/460-470 nm | | O1524684 | One-Step LumiDye™ 594 Phalloidin/Nucleiblue Double Staining Kit
| 5 mL | 30 mL | 590/617 nm | 345-355/460-470 nm | | O1524685 | One-Step LumiDye™ 633 Phalloidin/Nucleiblue Double Staining Kit
| 5 mL | 30 mL | 630/650 nm | 345-355/460-470 nm | | O1524686 | One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit
| 5 mL | 30 mL | 681/698 nm | 345-355/460-470 nm | | O1524687 | One-Step Rhodamine Phalloidin/Nucleiblue Double Staining Kit
| 5 mL | 30 mL | 546/575 nm | 345-355/460-470 nm |
Note: The usable times of the kit refer to the total number of microwells that can be tested for adherent cells via fluorescence microscopy in 96-well plates.
|
Precautions:
⁕ Phalloidin is toxic (LD₅₀ = 2 mg/kg body weight). Adequate personal protection must be worn during handling.
⁕ Single-staining control groups are recommended for experiments to adjust compensation in flow cytometry assays.
⁕ Fluorescent dyes contained in the kit require storage away from light.
⁕ Phalloidin staining is recommended to be performed after immunolabeling.
⁕ For liquid-cultured yeast, cells in logarithmic growth phase yield far better staining results than those in stationary phase.
⁕ This product is for research use only and shall not be stored in residential premises.
⁕ Follow standard laboratory safety protocols in your facility for your personal safety and health.
Instructions for Use:
I. Pre-experiment Preparation
1. Reagent Preparation
Allow all kit components to equilibrate to room temperature; prepare sufficient PBS buffer, 4% paraformaldehyde and Triton X-100.
2. Instrument Preparation
Fluorescence microscope: Excitation/Emission wavelengths (Ex/Em): 490/515 nm, 555/565 nm, 590/617 nm, 630/650 nm, 681/698 nm, 546/575 nm, 345–355/460–470 nm.
Flow cytometer: Detection channel Excitation/Emission wavelengths (Ex/Em): 490/515 nm, 555/565 nm, 590/617 nm, 630/650 nm, 681/698 nm, 546/575 nm, 345–355/460–470 nm.
3. Cell Preparation
Treat cells with corresponding drugs in accordance with your experimental design.
II. Operating Procedures
Protocol 1: Fluorescence Microscopy Detection (for Suspension Cells)
1. Cell Collection and Washing
(1) Transfer the suspension cell sample to be tested into a centrifuge tube.
(2) Centrifuge at 1000 rpm for 5 minutes at room temperature, then carefully aspirate and discard the supernatant.
2. Cell Resuspension and Counting
(1) Resuspend cells with PBS and perform cell counting.
(2) Transfer 5×10⁵ cells into a new centrifuge tube, centrifuge at low speed of 1000 rpm for 5 minutes, and remove PBS.
Note: Cell density is not strictly restricted for microscopy detection; only ensure consistent cell density between experimental and control groups. Adjust flexibly according to sample type and experimental conditions to achieve clear, distinguishable cell distribution under the microscope field of view.
3. Cell Fixation
(1) Add an appropriate volume of 4% paraformaldehyde fixative, fix cells at room temperature for 15 min, then wash with PBS 2–3 times.
Note: Methanol and other organic solvents will disrupt actin structures during fixation. Methanol-free paraformaldehyde is recommended as the fixative.
4. Cell Permeabilization
(1) (Optional) Permeabilize cells with 0.4% Triton X-100 solution prepared in PBS at room temperature for 15 min to enhance membrane permeability.
(2) Wash with PBS 2–3 times.
Note: Higher centrifugation speed can be adopted when collecting fixed and permeabilized cells.
5. Cell Staining
(1) Add 500 μL of staining working solution (One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit).
Sample Quantity
| 1 | 5 | 10 | One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit
| 500 μL
| 2500 μL
| 5000 μL |
|
(2) Incubate for 15 minutes at room temperature protected from light (incubation time may be optimized based on preliminary trial results).
Note: Incubation duration and loading volume of the dye can be adjusted according to different cell types, preliminary experimental data, or customized dosages set by experimental conditions.
6. Washing
(1) Aspirate and discard the staining working solution after incubation completes.
(2) Wash the cells with PBS 2–3 times.
7. Microscopic Observation and Imaging
(1) 96-well plate method: Add 100 μL cell suspension into a 96-well plate, let stand for a short while until cells settle naturally to the bottom, then observe under a fluorescence microscope.
Note: The volume of cell suspension can be adjusted according to cell counts; optimal cell confluency in the microscope field is 70%–80%.
(2) Glass slide method: Pipette 20–30 μL cell suspension onto a clean glass slide, gently place a coverslip to avoid air bubbles, then observe under a fluorescence microscope.
(3) Locate target fields and capture images separately under preset filter sets.
Protocol 2: Fluorescence Microscopy Detection (for Adherent Cells)
1. Cell Treatment
(1) Seed cells into well plates one day in advance to reach 70%–85% confluence. After cells adhere fully, treat them following your experimental design.
2. Cell Washing
(1) Aspirate cell culture medium directly from the culture plate.
(2) Gently wash cells 2–3 times with PBS at volume matched to plate format (e.g., 100 μL per well for 96-well plates, 150 μL per well for 48-well plates, 250 μL per well for 24-well plates, 500 μL per well for 12-well plates, 1 mL per well for 6-well plates). Aspirate all PBS after washing.
3. Cell Fixation
(1) Add an appropriate volume of 4% paraformaldehyde fixative, fix cells at room temperature for 15 min, then rinse with PBS 2–3 times.
Note: Methanol and other organic solvents disrupt actin structures during fixation. Methanol-free paraformaldehyde is recommended as fixative.
4. Cell Permeabilization
(1) (Optional) Permeabilize cells with 0.4% Triton X-100 in PBS at room temperature for 15 min to boost membrane permeability.
(2) Rinse with PBS 2–3 times.
Cell Staining
(1) Add staining working solution (One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit) to each well at the same volume specified in Step 2 (e.g., 100 μL/well for 96-well, 150 μL/well for 48-well, 250 μL/well for 24-well, 500 μL/well for 12-well, 1 mL/well for 6-well plates).
Sample Quantity (96-well Plate)
| 1 | 5 | 10 | One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit
| 100 μL
| 500 μL
| 1000 μL |
|
(2) Incubate for 15 minutes at room temperature in the dark (incubation time can be optimized based on preliminary experimental results).
Note: The incubation duration and loading volume of the dye can be adjusted according to different cell types, preliminary test data, or customized dosages for experimental conditions.
6. Washing
(1) Aspirate the staining working solution from each well once incubation is finished.
(2) Wash the cells 2–3 times with PBS at the same volume indicated in Step 2.
7. Microscopic Observation and Image Capture
(1) The labeled phalloidin exhibits excellent photostability. Samples can be imaged directly in PBS; for optimal performance, an anti-fade mounting medium is also applicable.
Note: Imaging immediately is strongly recommended if samples are not mounted. For best outcomes, stained samples should be mounted with suitable mounting medium and stored at 4 °C protected from light.
Protocol 3: Flow Cytometry Detection (for Suspension and Adherent Cells)
1. Collection and Preparation of Single-Cell Suspension
⁕ Suspension cells: Follow the procedures in Protocol 1; finally collect 4×10⁵–6×10⁵ cells.
⁕ Adherent cells:
a. Digest cells with an appropriate amount of trypsin digestion solution.
Note: The dosage of trypsin, digestion duration and temperature shall be operated per laboratory routine experience.
b. Key step: Add complete cell culture medium to terminate digestion.
c. Transfer cell suspension into a suitable centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, then aspirate and discard supernatant.
d. Resuspend cells with PBS and conduct cell counting.
e. Take 4×10⁵–6×10⁵ cells, centrifuge at 1000 rpm for 5 min at room temperature, remove supernatant.
f. Wash cells with PBS, centrifuge at 1000 rpm for 5 min at room temperature, discard supernatant.
2. Cell Fixation
(1) Add appropriate volume of 4% paraformaldehyde fixative, fix cells at room temperature for 15 min, then wash with PBS 2–3 times.
Note: Methanol and other organic solvents will disrupt actin structures during fixation. Methanol-free paraformaldehyde is recommended as fixative.
3. Cell Permeabilization
(1) (Optional) Permeabilize cells with 0.4% Triton X-100 diluted in PBS at room temperature for 15 min to enhance membrane permeability.
(2) Rinse with PBS 2–3 times.
Note: Higher centrifugation speed can be used when harvesting fixed and permeabilized cells.
4. Cell Staining
(1) Add 0.5 mL staining working solution to resuspend into single-cell suspension. Incubate for 15 min at room temperature away from light.
Note: Washing with PBS 1–3 times is also feasible after incubation.
Sample Quantity
| 1 | 5 | 10 | One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit
| 500 μL
| 2500 μL
| 5000 μL |
Note: The incubation time and loading volume of the dye can be adjusted according to different cell types used in the experiment, preliminary experimental results or customized dosages set for experimental conditions. |
5. Detection
(1) Important: Perform flow cytometry detection within 1 hour after staining to ensure stable fluorescent signals.
(2) Analyze samples via flow cytometer under preset detection channels.
(3) Adjust voltage using blank control (unstained cells) first to place cell populations at the lower-left quadrant of the coordinate system.
(4) Subsequently test negative and positive controls to verify valid experimental system.
(5) Finally test experimental samples, record and analyze the mean fluorescence intensity of cells.
Protocol 4: Fluorescence Microscopy Detection (for Tissue Sections)
1. Section Washing
(1) Rinse tissue sections twice with PBS.
2. Fixation
(1) Fix tissue sections or cells with 3.7% formaldehyde solution prepared in PBS at room temperature for approximately 10–20 min.
Note: Methanol destroys actin structure; fixatives containing methanol are prohibited, and methanol-free formaldehyde is strongly recommended.
(2) Wash 2–4 times with PBS containing 0.1% Triton X-100, 5 minutes per wash.
3. Staining and Washing
(1) Drop staining working solution onto each section at a volume of 200 µL per slide, incubate for 30–60 min at room temperature protected from light. Place slides in a slide staining box during incubation to prevent liquid evaporation.
Sample Quantity
| 1 | 5 | 10 | One-Step LumiDye™ 680 Phalloidin/Nucleiblue Double Staining Kit
| 200 μL
| 1000 μL
| 2000 μL |
Note: The incubation time and volume of the dye applied can be adjusted according to different types of tissue sections, preliminary experimental results, or customized dosages specified for experimental conditions. |
(2) Wash 2–4 times with PBS containing 0.1% Triton X-100, 5 minutes each time.
4. Microscopic Observation and Image Capture
(1) Samples can then be directly observed under a fluorescence microscope.
Note: For long-term preservation, mount the slides after air-drying naturally and store at 4 °C away from light; the preservation period can reach approximately 6 months.
III. Result Interpretation
1. Qualitative Analysis (Microscopy)
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Figure 1. Fluorescence micrograph of fixed and permeabilized HeLa cells co-stained with LumiDye™ 680 Phalloidin and Hoechst 33342
After staining incubation at room temperature, LumiDye™680 Phalloidin specifically binds to cellular microfilaments (F-actin) in frozen mouse lung sections, presenting intact microfilament fibers with distinct and orderly arrangement. Nucleiblue targets and labels cell nuclei with regular nuclear morphology. The merged image shows clearly divided signal regions, with negligible non-specific staining and fluorescence crosstalk, and balanced signal intensity for dual staining.
Note: Actual test results may vary depending on detection equipment, operating conditions, cell types and other factors. This example is for reference only.
Frequently Asked Questions:
1. Q: Can methanol be used for cell fixation during phalloidin cytoskeleton staining?
A: Methanol degrades actin protein. Fixatives containing methanol must not be used; methanol-free paraformaldehyde is required.
2. Q: Can this product be used for staining paraffin sections?
A: No. Embedding agents in paraffin sections damage F-actin, which will cause poor or absent staining signals.
3. Q: Cytoskeleton staining is partially successful in fixed live cell samples without permeabilization. What causes this partial staining?
A: Cells fixed with 4% paraformaldehyde maintain intact cellular morphology and structure, yet the lipolytic property of the fixative modifies cell membrane permeability, allowing LumiDye™-conjugated phalloidin to penetrate cells and bind F-actin. However, the lack of Triton X-100 permeabilization limits uniform penetration, resulting in only partial cell staining. The solution is to perform both fixation and permeabilization procedures.
