Autophagy/Cytotoxicity Dual Detection Kit (MDC/PI Method) provides a convenient tool for studying the regulatory mechanisms of autophagy and cytotoxicity at the cellular level. Using monodansylcadaverine (MDC) as an autophagy probe and propidium iodide (PI) as a cell death marker, this kit enables simultaneous analysis of both indicators.
Autophagy is a vital physiological process in which cells degrade and digest intracellular components (such as organelles and proteins) via lysosomes. This process not only allows cells to efficiently mobilize and recycle their own constituents, but also prevents the accumulation of damaged organelles, misfolded proteins, and invasive microorganisms. Autophagy proceeds through multiple steps: first, cytoplasmic components to be degraded are enclosed by a double-membrane structure to form autophagosomes; autophagosomes then fuse with lysosomes to form autolysosomes, where intracellular materials are ultimately degraded.
Regarding the detection principle: as a fluorescent compound, MDC interacts with membrane lipids through an ion-trapping mechanism and specifically accumulates in autophagosomes, emitting green fluorescence for the visual detection of autophagy. Meanwhile, PI in the kit is used for cytotoxicity analysis. PI stains necrotic cells and late apoptotic cells with impaired membrane integrity, causing these cells to emit red fluorescence. Note that MDC may also label other acidic organelles, and normal cells exhibit basal background fluorescence. Therefore, control groups should be included in result analysis to ensure accuracy.
Normal autophagy is essential for cell survival, differentiation, development, and homeostasis, while dysregulated autophagy is closely associated with cancer, infectious diseases, aging, and degenerative diseases. Notably, autophagy generally promotes cell survival under stress, but may also induce cell death under specific conditions. The relationship between autophagy and apoptosis is highly complex: they share common stimuli and components, and can mutually regulate each other’s activity. Thus, in-depth research into the factors and regulatory mechanisms governing cell fate decisions between autophagy and apoptosis is of great scientific significance.
Note: Performance equivalent to Abcam Autophagy/Cytotoxicity Dual Staining Kit (ab133075).
Applications
Analysis of cellular autophagy and cytotoxicity under conditions including drugs, nutrients, hypoxia, and oxidative stress.
Product Features
1. Dual simultaneous detection: Autophagy and cytotoxicity can be measured in one assay, facilitating investigation of their relationship.
2. Multi-instrument compatible: Stained samples can be observed in real time by fluorescence microscopy for morphology, or quantitatively analyzed by microplate reader and flow cytometry to meet diverse detection requirements.
3. Physiological relevance: MDC has low cytotoxicity, allowing detection closer to physiological autophagy levels and avoiding structural damage from fixation or permeabilization.
4. Rapid assay: No complicated pretreatment; staining is completed in 10 minutes with a simple procedure, saving experimental time.
5. Easy operation: No transfection required; direct staining of living cells without transfection or stable cell line construction.
6. Intuitive results: Clear distinction by fluorescence color for easy observation, with support for quantitative analysis and interpretable data.
Product Parameters
1. MDC Stain Ex/Em: 335/512 nm
2. PI Ex/Em: 493/636 nm (free, without DNA)
3. PI Ex/Em: 535/617 nm (DNA-bound)
Kit Components
| C1520245 | Component | 100 T | 10×100 T | Storage | | C1520245A | 10× MDC Stain | 1 mL | 10×1 mL | -20℃. Store in the dark. | | C1520245B | Stain Buffer | 50 mL | 10×50 mL | 2-8℃. | | C1520245C | Wash Buffer | 100 mL | 10×100 mL | 2-8℃. | | C1520245D | PI | 500 μL | 10×500 μL | -20℃. Store in the dark. |
|
Note: 100 T refers to the total number of wells detectable for adherent cells using Component A (10× MDC Stain) in a 96-well plate under fluorescence microscopy.
Precautions
1. Fluorescent dyes are susceptible to quenching upon light exposure. Please perform all operations in the dark to maintain dye stability.
2. The dyes are potentially harmful to humans. Personnel must wear lab coats and disposable gloves during operation. Additional protective equipment such as goggles may be worn if necessary to avoid direct contact of reagents with skin, mucous membranes and eyes.
3. Keep away from light during operation and minimize the time that all reagents are exposed to strong light.
4. Normal cell control groups can be set up in the experiment to compare and analyze the fluorescence intensity between the drug-treated group and the control group, so as to determine whether autophagy is induced or inhibited. Meanwhile, interference from non-specific staining should be excluded, and results should be further verified in combination with autophagy-related indicators.
5. This product is for research use only and must not be stored in ordinary residential premises.
6. For your safety and health, please follow the standard laboratory safety regulations of your institution.
Instructions for Use
I. Pre-Experiment Preparation
1. Reagent Preparation
Remove Component A (10× MDC Stain), Component B (Stain Buffer), Component C (Wash Buffer), and Component D (PI) from storage (e.g., -20 °C/4 °C) and equilibrate to room temperature (15-25 °C). Prepare an adequate volume of PBS buffer.
2. Instrument Preparation
Fluorescence microscope:
For MDC detection: Use an ultraviolet excitation filter (330–350 nm) and an emission filter (510–530 nm) to detect green fluorescence.
For PI: After binding to DNA, the maximum excitation wavelength is 535 nm and the maximum emission wavelength is 617 nm.
3. Control Group Setup
| Group | MDC | PI | Sample Cell | | Blank Control | - | - | Untreated cells | | Negative Control | + | + | Untreated cells | | Positive Control | + | + | Autophagy-induced cells | | Experimental Group | + | + | Experimentally treated cells |
|
(1) Blank Control: Unstained cells, used to assess cellular autofluorescence.
(2) Negative Control: Stained cells without autophagy induction, representing the basal autophagy level.
(3) Positive Control: Induce autophagy by starving cells in EBSS (Earle’s Balanced Salt Solution). For initial experiments, incubate at 37 °C for 30 min to 4 h with a time gradient to determine the optimal induction duration.
(4) Experimental Group: Treat cells in advance according to the experimental design (e.g., drug treatment, starvation, etc.).
Note: EBSS lacks amino acids and serum, mimicking a "starvation state"-it is a commonly used positive control induction method in autophagy research.
Note: Sensitivity to autophagy induction varies across cell lines; optimize the treatment duration for your specific cell type. For example, some non-neuronal cells (e.g., HeLa, HEK293) show autophagy activation after 30 min of EBSS treatment, but the exact response time should be evaluated using your experimental detection method.
II. Procedure
Protocol A: Fluorescence Microscopy (For Suspension Cells)
3. Cell Resuspension and Counting
(1) Add an appropriate volume of Component C (Stain Buffer) to resuspend the cells and perform cell counting.
(2) Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 min at low speed, and remove Component C.
(3) Resuspend in 100 μL of Component B (Wash Buffer). Centrifuge at 1000 rpm for 5 min at low speed and discard the supernatant.
Note: The microscopy method is not strict with cell density, as long as the density is consistent between the experimental group and the control group. It can be flexibly adjusted according to sample type and experimental conditions, with a recommended cell coverage of 70%-85% under the microscope.
Note: Component B (Stain Buffer) is an independently developed staining buffer optimized through multiple formulations. It maintains cell physiological status and provides basic nutrition during detection, while significantly improving dye stability. If volume is insufficient due to sample differences, PBS, HBSS or other conventional buffers may be used to adjust the volume for resuspension without affecting results.
4. Cell Staining
(1) Prepare MDC/PI staining working solution at a ratio of Component A (10× MDC Stain): Component B (Stain Buffer): Component D (PI)=2: 17: 1, then add the working solution to the cells. (Prepare fresh before use and protect from light during preparation.) See Table 1 for working solution preparation.
Table 1. Preparation of MDC/PI Staining Working Solution
(For a 100 μL staining system per well in a 96‑well plate, prepare MDC/PI staining working solution according to the table below and mix thoroughly.)
| Reaction Component | 1 Sample | 5 Samples | | A. 10× MDC Stain | 10 μL | 50 μL | | B. Stain Buffer | 85 μL | 425 μL | | D. PI | 5 μL | 25 μL | | Total Volume | 100 μL | 500 μL |
|
Note: The volume of the reaction mixture can be scaled proportionally based on actual experimental needs.
(2) After adding 100 μL of MDC/PI staining working solution to each well, incubate at 37 ºC for 15 min in the dark. The incubation time can be flexibly adjusted from 10 to 60 min according to the actual staining effect observed.
5. Washing and Resuspension
(1) After incubation, centrifuge at 1000 rpm for 5 min at room temperature to collect the cells.
(2) Carefully aspirate and discard the dye-containing supernatant (this is waste liquid and should be disposed of properly).
(3) Add 100 μL of Component C and gently resuspend the cells for washing. Repeat this washing step 3 times to sufficiently remove unbound free dye and reduce background.
(4) After the final wash, completely aspirate the supernatant, add 100 μL of Component C to resuspend the cells, and prepare the sample for testing.
6. Microscopic Observation and Imaging
(1) 96-well plate method: Add 100 μL of cell suspension to a 96-well plate, let stand briefly until the cells naturally settle to the bottom, then observe under a fluorescence microscope.
(2) Slide method: Pipette 20-30 μL of cell suspension onto a clean glass slide, gently cover with a coverslip to avoid air bubbles, and observe under a fluorescence microscope.
(3) Under preset filter conditions, locate and capture green punctate or vesicular fluorescence (i.e., autophagosomes/autolysosomes).
Note: The volume of cell suspension can be adjusted according to cell count. Optimal cell distribution in the microscope field is 70%-85%.
Note: Samples must be analyzed immediately after staining. It is recommended to detect MDC staining first, followed by PI staining, to avoid depletion of MDC fluorescence during analysis.
Protocol B: Fluorescence Microscopy (For Adherent Cells)
1. Cell Treatment
Inoculate cells into a plate one day in advance to reach 70%-85% confluence. After cell attachment, treat cells according to the experimental design.
2. Cell Washing
(1) Aspirate and discard the cell culture medium directly from the culture plate.
(2) Add an appropriate volume of Component C (Wash Buffer) according to the plate format to gently wash the cells once (e.g., 100 μL/well for 96-well plates, 150 μL/well for 48-well plates, 250 μL/well for 24-well plates, 500 μL/well for 12-well plates, 1 mL/well for 6-well plates). Completely aspirate Component C after washing.
3. Preparation of Staining Working SolutionBased on the number of sample wells, prepare MDC/PI staining working solution in advance at a ratio of Component A (10× MDC Stain): Component B (Stain Buffer): Component D (PI)=2: 17: 1 under dark conditions (prepare fresh before use), then add the working solution to the cells. See Table 1 above for preparation.
4. Cell Staining
(1) Add the corresponding volume of MDC/PI staining working solution to each well (same volume as in Step 2, e.g., 100 μL/well for 96-well plates, 150 μL/well for 48-well plates, 250 μL/well for 24-well plates, 500 μL/well for 12-well plates, 1 mL/well for 6-well plates).
(2) After adding 100 μL of MDC/PI staining working solution to each well, incubate at 37 ºC for 15 min in the dark. The incubation time can be flexibly adjusted from 10 to 60 min according to the actual staining effect observed.
5. Washing
(1) After incubation, aspirate and discard the staining working solution from each well.
(2) Add Component C to wash the cells at the same volume as in Step 1. Repeat this washing step 3 times, and completely aspirate the wash buffer each time.
6. Microscopic Observation and Imaging
(1) After the final wash, add the corresponding volume of Component C to each well to keep the cells moist.
(2) Observe and image the culture plate directly under a fluorescence microscope. Filter settings are the same as in Protocol A.
Note: Samples must be analyzed immediately after staining. It is recommended to detect MDC staining first, followed by PI staining, to avoid depletion of MDC fluorescence during analysis.
III. Result Interpretation
Qualitative Analysis (Microscopy):
Figure 1. MDC Staining Image
(1) To detect whether ZnMet can induce more autophagosomes in fibroblasts, a blank control group, Met group, ZnCl₂ group, and ZnMet group were set up to exclude interference from single components. After incubation with MDC probe, fluorescence images were captured using a Nikon confocal A1R system. The results indicated that ZnMet can activate the autophagy pathway.
Note: Image reproduced from A novel sprayable thermosensitive hydrogel coupled with zinc modified metformin promotes the healing of skin wound. Bioact Mater. (doi: 10.1016/j.bioactmat.2022.06.008.)
Note: MDC is an acidic dye containing an amino group. MDC may also label other acidic organelles, and normal cells exhibit basal background fluorescence. Therefore, control groups must be included for result analysis.
(2) Result Interpretation (Recommended)
| Cell Status | Autophagy Probe Fluorescence (MDC, Green) | Cytotoxicity Probe Fluorescence (PI, Red) | Result Description | | Normal Viable Cells | Weak/No fluorescence | No fluorescence | Low autophagy level, intact cell membrane | | Autophagy-Activated Viable Cells | Strong fluorescence (punctate autophagosome distribution) | No fluorescence | High autophagy level, viable cells | | Dying/Dead Cells | Weak/No fluorescence | Strong fluorescence (diffuse cytoplasmic distribution) | Damaged cell membrane, positive cytotoxicity | | Autophagy-Activated Dying Cells | Strong fluorescence | Red fluorescence | Autophagy activated but cells damaged |
|
Frequently Asked Questions
1. Q: A large number of adherent cells detach after staining. How to solve this problem?
A: For cells with weak adherence, pre-treat the culture plate with poly-L-lysine, rat tail collagen, or gelatin in advance to enhance cell attachment to the plate. Add samples gently during operation to avoid direct impact on the cell layer. Tilt the plate slowly during washing and add washing buffer along the wall to reduce disturbance to cells by liquid flow.
2. Q: How to precisely select the MDC staining time (10-60 min)?
A: This is a parameter that requires optimization. Too short a time may result in weak signals, while too long a time may increase non-specific staining and background. Recommendation: Before formal experiments, perform a preliminary experiment using positive control cells (EBSS starvation treatment), e.g., 30 min, then adjust the staining time within 10-60 min according to the preliminary results. Select the time point with the highest signal-to-noise ratio (strongest positive signal, lowest background) via microscopy or flow cytometry as the standard staining time for subsequent experiments.
3. Q: Why wash 3 times in the microscopy protocol?
A: Microscopy involves direct imaging. Any residual free dye will produce uniform background fluorescence in the field of view, severely interfering with the observation of punctate autophagosome signals. Therefore, strict washing 3 times is required to maximize the signal-to-noise ratio.
4. Q: How to troubleshoot high staining intensity and strong background in the negative control?
A: High background in the negative control may result from non-standard procedures (e.g., insufficient washing times, excessive staining duration) or inappropriate positive control setup. Strictly follow the washing steps to ensure at least 3 washes after MDC staining to thoroughly remove residual dye. Meanwhile, verify instrument parameters to ensure excitation wavelength is 350–380 nm and emission wavelength is 512-532 nm. Set up positive controls (e.g., cells induced with EBSS for 30 min-4 h) and blank controls to verify the reliability of the negative control by comparison.
5. Q: Why is the expected fluorescence not observed? How to resolve it?
A: Failure to observe expected fluorescence may result from incorrect procedures, improper excitation light settings, or inappropriate positive control setup. Carefully verify correct excitation wavelength settings, strictly follow the experimental protocol, and confirm reasonable positive control setup (e.g., using EBSS-induced cells).
6. Q: What causes low fluorescence intensity of both MDC and PI staining?
A: Possible causes are excessively low cell density or cell loss during processing.
ZJ26F0535133