Autophagy Green Detection Kit (MDC Method) is a fluorescence staining kit for detecting cellular autophagy. This kit employs the fluorescent probe Monodansylcadaverine (MDC), an acidophilic dye that binds specifically to lipids on autophagosome membranes via ion-trapping effects. It rapidly and selectively accumulates in autophagic vesicles (autophagosomes) in living cells and emits bright green fluorescence, enabling direct visual evaluation of autophagic activity in in vitro cultured cell models. Compared with traditional autophagy detection methods, this kit has prominent advantages: no transfection is required, and rapid detection can be achieved by direct fluorescence staining; MDC shows high specificity for autophagosomes. Note: MDC may also label other acidic organelles, and normal cells exhibit basal background fluorescence. Therefore, control groups must be set for result analysis.
Autophagy is a highly evolutionarily conserved intracellular catabolic pathway. By forming double-membraned autophagosomes, it sequesters and delivers damaged organelles, misfolded proteins, and invasive pathogens to lysosomes for degradation and recycling. This pathway is not only a core mechanism for cells to obtain energy, maintain survival, differentiation, and homeostasis under adverse conditions such as starvation and oxidative stress, but also plays a vital role in organismal growth and development, immune responses, and environmental adaptation. Dysregulation of autophagy is closely associated with major pathological processes including tumors, neurodegenerative diseases, metabolic diseases, pathogen infections, and aging. Given its dual core roles in physiology and pathology, accurate and efficient monitoring of cellular autophagic activity has become an indispensable key step and cutting-edge hotspot in modern life science and medical research.
Application
Real-time observation of changes in autophagic activity in living cells;
Routine autophagy detection without a transfection platform;
Screening and semi-quantitative analysis of autophagic activity under conditions such as drugs, nutrients, hypoxia, and oxidative stress;
Combined studies with apoptosis, necrosis, cell cycle, and other assays;
Product Features
1. Rapid assay: Imaging can be performed after 10 minutes of staining, with results available on the same day, saving 1-2 days compared with traditional transfection methods.
2. Simple operation: No transfection required; direct staining of living cells without transfection or stable cell line construction.
3. Wide applicability: Suitable for hard-to-transfect cells such as primary cells.
4. Low-toxic real-time detection: In situ staining of living cells with MDC, no fixation/permeabilization damage, allowing continuous tracking of autophagic dynamics.
5. High sensitivity: Acidophilic MDC targets autophagosomes/lysosomes, with distinct punctate fluorescent signals.
Product Parameters
Ex/Em: 335 nm / 512 nm
Kit Components
| C1520234 | Component | 100 T | Storage | | C1520234A | 10× MDC Stain | 1 mL | -20℃. Store in the dark. | | C1520234B | Stain Buffer | 50 mL | 2-8℃. | | C1520234C | Wash Buffer | 100 mL | 2-8℃. |
|
Note: 100 T refers to the total number of wells detectable for adherent cells using Component A in a 96-well plate under fluorescence microscopy.
Precautions
1. MDC Stain is potentially toxic. 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 the reagent with skin, mucous membranes, and eyes.
2. Protect from light: MDC is highly light-sensitive. Minimize light exposure during all procedures and reduce the time reagents are exposed to strong light.
3. Set normal cell control groups to compare fluorescence intensities between drug-treated groups and controls to determine whether autophagy is induced or inhibited. Eliminate interference from non-specific staining and further validate results with autophagy-related markers.
4. This product is only for fluorescence staining detection of autophagy in cultured cells or tissues. It cannot be used for frozen cells/tissues or fixed cells/tissue sections.
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 general laboratory safety regulations.
Instructions for Use
I. Pre-Experiment Preparation
1. Reagent Preparation
Remove Component A (10× MDC Stain), Component B (Stain Buffer), and Component C (Wash Buffer) from storage (-20℃/4℃) and equilibrate to room temperature (15–25℃).
2. Instrument Preparation
Fluorescence microscope: excitation filter 330-350 nm (e.g., DAPI filter); emission filter 510–530 nm (e.g., FITC filter).
3. Cell Preparation
Treat cells accordingly based on experimental design (e.g., drug induction, starvation induction).
4. Control Group Setup
| Group | MDC | 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 for assessing cellular autofluorescence.
(2) Negative Control: Stained cells without autophagy induction, representing basal autophagy levels.
(3) Positive Control: Induce autophagy by starving cells in EBSS (Earle’s Balanced Salt Solution). For initial experiments, incubate at 37℃ for 30 min–4 h with a time gradient to determine optimal induction duration.
Note: EBSS lacks amino acids and serum, mimicking starvation; it is a standard positive control method in autophagy research. Sensitivity to autophagy induction varies by cell line. Optimize treatment duration accordingly. Some non-neuronal cells (e.g., HeLa, HEK293) show autophagy activation after 30 min of EBSS treatment; exact response time should be evaluated by detection method.
II. Protocol
Protocol A: Fluorescence Microscopy (Suspension Cells)
1. Cell collection and washing
(1) Collect suspension cells into centrifuge tubes.
(2) Centrifuge at 1000 rpm for 5 min at room temperature; carefully discard supernatant.
(3) Add appropriate Component C (Wash Buffer) and gently resuspend cells.
2. Resuspension and counting
Count cells, take 1×10⁵ cells, centrifuge at 1000 rpm for 5 min, remove supernatant.
Note: Cell density is flexible as long as test and control groups are consistent (recommended: 10⁵-10⁷ cells/mL). Adjust for clear cell distribution under microscopy.
Note: If Component B is insufficient, PBS or HBSS may be used without affecting results.
3. Cell staining
(1) Add 90 μL Component B and 10 μL Component A (final concentration 1×); mix gently.
(2) Incubate at 37℃ for 15 min in the dark (adjustable 10-60 min based on staining efficiency).
4. Washing and resuspension
(1) After incubation, centrifuge at 1000 rpm for 5 minutes at room temperature to harvest 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 (Wash Buffer) and gently resuspend the cells for washing. Repeat this washing step a total of 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.
5. 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 and attach 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 images of green punctate or vesicular fluorescence (i.e., autophagosomes/autolysosomes).
Protocol B: Fluorescence Microscopy (Adherent Cells)
1. 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 culture 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
2. Component C after washing.
Preparation of Staining Working SolutionPrepare MDC staining working solution at a ratio of Component A : Component B = 1:9 under light‑protected conditions in advance, based on the number of sample wells (prepare fresh before use). For example, to stain one 96‑well plate, prepare 10 mL of working solution (1 mL Component A + 9 mL Component B).
3. Cell Staining
(1) Add the corresponding volume of MDC staining working solution to each well (same volume as in Step 1; 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) Incubate at 37 °C for 15 min in the dark. The incubation duration can be flexibly adjusted from 10 to 60 min according to the actual staining performance observed.Note: The dosage of each dye can be adjusted according to different cell types, preliminary experiments, or experimental conditions.
4. Washing
(1) After incubation, aspirate and discard the staining working solution from each well.
(2) Add Component C (Wash Buffer) at the same volume as in Step 1 to wash the cells. Repeat this washing step 3 times, and completely aspirate the wash buffer each time.
5. 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.
III. Result Interpretation
Qualitative Analysis (Microscopy)
Figure 1. MDC Staining Image
To verify whether Ato can promote autophagy in OB‑6 cells, a blank control group, Dex group, and Dex+Ato group were set up to exclude interference from single components. Dex (1.0 μM) and Ato (0.1 μM) were used, followed by incubation with the MDC probe. Under a fluorescence microscope, green punctate or vesicular fluorescent signals in the cytoplasm represent autophagosomes or autolysosomes. The results indicate that Ato can promote cellular autophagy.
Note: Image reproduced from Atorvastatin Upregulates microRNA‑186 and Inhibits the TLR4‑Mediated MAPKs/NF‑κB Pathway to Relieve Steroid‑Induced Avascular Necrosis of the Femoral Head. Front Pharmacol (doi:10.3389/fphar.2021.583975.)
Note: MDC is an acidic dye containing an amino group. MDC may also label other acidic organelles, and normal cells exhibit basal background fluorescence. Control groups must be included for result analysis.
Frequently Asked Questions
Q1: Is trypsinization necessary for adherent cells? How long is considered “non‑excessive” trypsinization? Are there gentler alternatives?
A: Yes. Gentle agitation with 0.25% trypsin at room temperature for 30-60 seconds is sufficient for cell detachment. Digestion longer than 2 minutes may easily induce false‑positive stress autophagy.
a. Cell scraper: For firmly adherent cells, they can be gently scraped off on ice using a cell scraper and collected with buffer. This method is gentler but may produce cell clumps, which require pipetting or filtration (e.g., through a 40 μm cell strainer) to obtain a single‑cell suspension.
b. Non‑enzymatic cell dissociation solution: Commercially available dissociation solutions based on EDTA or special buffers can detach cells without proteases, cause less damage to cell surface structures, and are preferred for studying cellular processes such as autophagy.
Q2: How to resolve excessive cell detachment during staining and washing (especially in the adherent cell protocol)?
A: This usually occurs in cells with poor viability or weak adherence (e.g., after autophagy induction).
a. Pre‑coating: Treat culture plates or slides with poly‑L‑lysine, rat tail collagen, or gelatin before cell seeding to enhance cell attachment.
b. Gentle handling: All liquid addition and aspiration must be performed gently to avoid direct impact on the cell layer. Add buffer slowly along the well wall during washing, and keep the pipette tip away from the cell layer when aspirating.
Q3: How to precisely choose the MDC staining time (10-60 min)?
A: This is a parameter requiring optimization. Insufficient staining time may lead to weak signals, while excessive time may increase non‑specific staining and background. Recommendation: Before formal experiments, perform a time‑gradient assay using positive control cells (EBSS starvation treatment) (e.g., 30, 45, 60, 90, 120 min, 5-6 time points). 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.
Q4: Why only one wash in the flow cytometry protocol but three washes in the microscopy protocol?
A: Microscopy involves direct imaging. Any residual free dye produces uniform background fluorescence in the field of view, severely interfering with the observation of punctate autophagosome signals. Therefore, rigorous washing (3 times) is required to maximize the signal‑to‑noise ratio.Flow cytometry detects transient fluorescence signals from single cells passing through a laser. The instrument can exclude most free dye particles (which differ from cells in size and complexity) via gating. Thus, one wash is usually sufficient to remove most background while better preserving bound MDC fluorescence, with simpler and faster operation.
Q5: Is the 500 μL volume of Component C for resuspension before flow cytometry fixed?
A: No. This volume aims to achieve a suitable cell concentration for detection.
a. Too low concentration: Prolongs detection time and results in insufficient statistical data.
b. Too high concentration: Prone to clogging the flow cytometer tubing and increased cell coincidence, affecting data accuracy.
c. Recommendation: The final cell concentration after resuspension should be approximately 2-5×10⁵ cells/mL. Therefore, resuspension in 500 μL is appropriate for an initial cell count of 1×10⁵ (i.e., 2×10⁵ cells/mL). Increase the resuspension volume accordingly for larger cell quantities.
Q6: Can samples be fixed after MDC staining?
A: Aldehyde fixation is not recommended, as it quenches fluorescence. MDC is a hydrophobic probe that weakly binds to autophagosomal membrane lipids. Aldehyde fixatives cause membrane protein crosslinking and lipid rearrangement, displacing the dye and reducing fluorescence intensity by 30-70%.
a. Subsequent permeabilization (e.g., Triton X‑100) after fixation almost eliminates the signal.
b. Improper pH and osmotic pressure of fixative may cause swelling or rupture of autophagic vacuoles and morphological distortion.
c. If fixation is necessary, 2-4% paraformaldehyde (PFA) prepared in PBS at pH 7.2-7.4 is recommended; fix at room temperature for ≤15 min; wash immediately 3 times with PBS after fixation to reduce residual free aldehyde.
d. Note: Perform detection or imaging as soon as possible (within 1 hour) after fixation to avoid further fluorescence quenching.
Q7: What causes overly strong or weak staining, and how to adjust?
A: Overly strong staining usually results from excessively long MDC incubation, leading to non‑specific binding to intracellular acidic structures (e.g., lysosomes). Appropriately shorten the staining time and strictly control incubation duration to avoid overtime.Weak staining may be caused by insufficient staining time or quenching/degradation of MDC stain due to light exposure or prolonged storage (MDC is highly light‑sensitive and prone to decomposition). Appropriately prolong staining time; prepare working solution fresh immediately before use and protect from light throughout preparation and storage; replace the kit if it has expired or shows color changes.
Q8: 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. Verify instrument parameters: excitation wavelength set to 335 nm (tolerance ±10 nm), emission wavelength set to 512 nm (tolerance ±10 nm). If 335 nm excitation is unavailable, 355 nm may be used as an alternative, with a slight expected increase in background. Include positive controls (e.g., cells induced with EBSS for 30 min-4 h) and blank controls to verify the reliability of the negative control via comparison.
Q9: Why is the expected fluorescence not observed, and 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).
ZJ26F0434285