This kit is an integrated cellular status detection tool. It adopts a multi-parameter fluorescent labeling strategy including phosphatidylserine (PS) externalization, membrane integrity assessment and total nuclear staining, to realize simultaneous differentiation and quantitative analysis of viable, apoptotic and necrotic cells. Its complementary and clear detection principles are as follows:
(1) Targeting the phosphatidylserine (PS) externalization characteristic of apoptotic cells, Annexin V is labeled with high-brightness R-PE. It specifically binds to PS exposed on the cell surface in a calcium-dependent manner, enabling apoptotic cells to exhibit red fluorescence.
(2) According to differences in cell membrane integrity, damaged membranes of necrotic and late apoptotic cells allow high-charge nucleic acid probe EthD Gold to penetrate and bind DNA, producing red fluorescence. Alternative dyes including Draq 7 for basic research are also available.
(3) Hoechst 33342 blue dye penetrates all cells universally and stains cell nuclei, serving as a stable internal reference for cell counting and quantitative calibration.
Compared with traditional single-label kits, this product has outstanding advantages. Multi-parameter simultaneous detection avoids errors caused by batch sample processing. Quadrant analysis by flow cytometry clearly distinguishes cell subsets: Q3: viable cells, Q4: early apoptotic cells, Q1: late apoptotic cells, Q2: necrotic cells. It is also compatible with morphological observation under fluorescence microscope, especially suitable for dynamic tracing of apoptotic progression and quantifying the proportion of apoptotic and necrotic cells in total cell population. Applications include basic research on cellular physiological status, efficacy evaluation of anticancer drugs and antimicrobial peptides, mechanism analysis of tumor apoptosis, and auxiliary detection of cellular status in clinical samples.
Apoptosis and necrosis are two distinct modes of cell death. Accurate discrimination between them is irreplaceable for life science research and clinical translation. Apoptosis is an active and orderly programmed cell death process involved in embryonic development, tissue homeostasis maintenance and immune clearance. Abnormal activation or inhibition of apoptosis is closely associated with cancers, Alzheimer’s disease and other critical illnesses. Necrosis is triggered by acute injury accompanied by inflammatory response, acting as a core pathological feature of sepsis, myocardial infarction and other diseases. Viable cell status directly reflects metabolic function and signal pathway integrity, serving as a key indicator for drug screening. Traditional single-label assays cannot simultaneously obtain quantitative data of three cell types, limiting research efficiency. This kit breaks such limitations. With synergistic multi-parameter detection, it achieves standardized and accurate cellular status analysis in one system. It provides reliable tools for revealing molecular mechanisms of apoptosis-necrosis imbalance (such as mitochondrial pathway regulation), supports quantitative assessment of drug-induced cell death efficiency, and offers technical support for clinical sample detection including tumor prognosis and infectious disease diagnosis, facilitating efficient translation from basic research to clinical application.
Applications
Detection of viable, apoptotic and necrotic cells in adherent and suspension cells; tumor research, drug toxicity assessment, stem cell differentiation monitoring. Particularly suitable for dynamic tracing of apoptotic processes such as chemotherapy-induced cell death patterns.
Product Features
1. Simultaneous detection of full cell status: One experiment distinguishes viable, apoptotic and necrotic cells, overcoming limitations of traditional single labeling and realizing three-in-one accurate classification.
2. Time-saving and high efficiency: Total staining procedure only takes 15 minutes, much faster than conventional multi-step protocols, greatly improving experimental throughput.
3. Excellent fluorescence intensity: LumiDye™ 488 possesses high quantum yield and superior photostability, ensuring clear apoptotic cell signals.
4. Simple operation: Multi-parameter detection is completed by one-step staining without complicated pretreatment. Results can be directly quantified via flow scatter plots or microscopic images.
Product Specifications
1. Annexin V-R-PE Ex/Em: 488/578 nm；
2. Draq 7 Ex/Em: 599~644/678~697 nm；
3. Hoechst 33342 Ex/Em: 350/461 nm (with DNA)。
Kit Components
Item No.
| Appearance | Components | 10 T | 50 T | 100 T | Storage | | E1520241A | Liquid | 1× Annexin V Binding Buffer | 5 mL | 50 mL×2 | 50 mL×4 | 2-8℃. Store in the dark. | | E1520241B | Liquid | Annexin V-R-PE | 50 μL | 250 μL | 500 μL | 2-8℃. Store in the dark. | | E1520241C | Liquid | Draq 7 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. | | E1520241D | Liquid | Hoechst 33342 | 10 μL | 50 μL | 100 μL | 2-8℃. Store in the dark. |
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Note: The number of uses of the kit refers to the total number of microwells that can be tested when performing fluorescence microscopy assays with a 96-well plate.
Precautions
1. Unfixed live cells are required for this assay. Annexin V-R-PE rely on intact cell membranes to differentiate viable, apoptotic and necrotic cells.
2. Single-staining control groups are recommended for fluorescence compensation adjustment in flow cytometry.
3. For research use only. Do not store this product in residential environments.
4. Comply with routine laboratory safety regulations to protect personal safety and health.
Instructions for Use
I. Pre-experiment Preparation
1. Reagent PreparationEquilibrate all kit components to room temperature (15–25 °C) and prepare sufficient PBS buffer.
2. Instrument Preparation
(1) Fluorescence microscope: Excitation/Emission wavelength Ex/Em: 488/578 nm, Ex/Em: 599~644/678~697 nm, Ex/Em: 350/461 nm;
(2) Flow cytometer: Detection channel excitation/emission wavelength Ex/Em: 488/578 nm, Ex/Em: 599~644/678~697 nm, Ex/Em: 350/461 nm.
3. Control Group Setup
| Group | Annexin V-R-PE
| Draq 7
| Hoechst 33342 | Sample Cells | | Blank Control | - | - | - | Untreated cells | | Negative Control | + | + | + | Untreated cells | | Single Positive Control 1 | + | - | - | Cells with obvious apoptosis after inducer treatment | | Single Positive Control 2 | - | + | - | Cells with obvious apoptosis after inducer treatment | | Single Positive Control 3 | - | - | + | Cells with obvious apoptosis after inducer treatment | | Test Group | + | + | + | Test sample cells |
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(1) Blank control: Evaluate background fluorescence and cellular autofluorescence, adjust threshold and voltage in flow cytometry.
(2) Negative control: Eliminate non-specific dye binding and verify staining specificity.
(3) Single-positive control: Avoid fluorescence spillover, calibrate signal intensity and adjust fluorescence compensation.
4. Cell Preparation
Treat cells with corresponding drugs according to experimental design.
II. Assay Procedures
Protocol 1: Fluorescence Microscopy (Suspension Cells)
1. Cell Collection and Washing
(1) Transfer suspended cell suspension into centrifuge tubes.
(2) Centrifuge at 1000 rpm for 5 min at room temperature and carefully discard supernatant.
(3) Resuspend cells gently with 100 μL PBS and wash.
(4) Centrifuge again at 1000 rpm for 5 min at room temperature and completely remove supernatant.
2. Cell Resuspension and Counting
(1) Resuspend cells with PBS and perform cell counting.
(2) Take 5×10⁴–1×10⁵ cells, centrifuge at low speed 1000 rpm for 5 min and remove PBS.
(3) Resuspend cells in 100 μL Buffer A (1×Annexin V Binding Buffer), centrifuge and discard supernatant.
Note: Cell density is flexible for microscopic detection as long as groups are consistent (recommended: 10⁵–10⁷ cells/mL). Adjust appropriately to ensure clear cell distribution under microscope.
3. cell Staining
(1) To 100 μL Buffer A-resuspended cells, add 5 μL Buffer B (Annexin V-R-PE), 1 μL Buffer C (Draq 7) and 1 μL Buffer D (Hoechst 33342). Mix gently by pipetting.
(2) Incubate for 15–30 min at room temperature in dark. Incubation time can be optimized via preliminary tests.
4. Washing and Resuspension
(1) After incubation, centrifuge cells at 1000 rpm for 5 min at room temperature.
(2) Discard dye-containing supernatant properly as hazardous waste.
(3) Wash cells with 100 μL Buffer A.
(4) After final washing, remove supernatant completely and resuspend cells in 100 μL Buffer A for detection.
5. Microscopic Observation and Imaging
(1) 96-well plate method: Add 100 μL cell suspension into wells, let cells settle naturally, then observe under fluorescence microscope.
(2) Slide method: Drop 20–30 μL cell suspension onto clean slides, cover coverslips carefully to avoid bubbles.
(3) Capture images under corresponding filter channels:
Annexin V-R-PE for early apoptosis detection: Ex/Em: 488/578 nm;
DRAQ7 for necrotic cell detection: Ex/Em: 599~644/678~697 nm;
Hoechst 33342 for viable cell detection: Ex/Em: 350/461 nm.
Protocol 2: Fluorescence Microscopy (Adherent Cells)
1. Cell Washing
(1) Discard culture medium directly from culture plates.
(2) Wash cells with Buffer A at appropriate volume: 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. Remove Buffer A after washing.
2. Prepare Staining Working Solution
According to well quantity, mix Buffer A with dyes freshly before use: 5 μL Buffer B, 1 μL Buffer C, 1 μL Buffer D per 100 μL Buffer A.Example: Staining one 96-well plate requires 10.7 mL working solution (10 mL A + 500 μL B + 100 μL C + 100 μL D).
3. Cell Staining
(1) Add an appropriate volume of staining working solution to each well (same volume as Step 1 (2): 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 for 15–30 minutes at room temperature in the dark. The incubation time can be optimized according to preliminary experimental results.
4. Washing
(1) After incubation, aspirate and discard the staining working solution in each well.
(2) Add Component A (1× Annexin V Binding Buffer) to wash cells at the volume specified in Step 1 (2). Aspirate the washing buffer after rinsing.
5. Microscopic Observation and Imaging
(1) After washing, add an appropriate volume of Component A to each well to keep cells moist.
(2) Place the culture plate directly under a fluorescence microscope for observation and image capture. The filter setting is the same as Protocol Ⅰ.
Annexin V-R-PE detects early apoptotic signals: Ex/Em: 488/578 nm;
DRAQ7 detects necrotic cell signals: Ex/Em: 599~644/678~697 nm;
Hoechst 33342 detects viable cell signals: Ex/Em: 350/461 nm.
Protocol 3: Flow Cytometry (Suspension & Adherent Cells)
1. Single Cell Suspension Preparation
(1) Suspension cells: Follow Protocol 1 Steps 1–2, collect 5×10⁵–1×10⁶ cells.
(2) Adherent cells:
a. Aspirate the cell culture medium into a suitable centrifuge tube for later use. Rinse adherent cells once with a small amount of PBS; aspirate the rinsed PBS and transfer it into the same centrifuge tube containing the collected medium. Centrifuge at 1000 rpm for 5 minutes at room temperature.
b. Add an appropriate amount of EDTA-free trypsin digestion solution (just enough to cover the cells). Observe under a microscope at room temperature. When the cells become rounded and intercellular gaps widen, gently pipette up and down to completely detach the cells.
Note: Trypsin digestion is critical for adherent cells. Insufficient digestion time requires vigorous pipetting to detach cells, which may cause mechanical damage and lead to false-positive apoptosis results. Excessively long digestion time also induces false-positive apoptosis, affecting the binding of phosphatidylserine (PS) on the cell membrane to Annexin V and interfering with apoptosis detection.
c. Key step: Add the reserved cell culture medium from step a to terminate digestion.
Note: The reserved culture medium in the centrifuge tube must not be discarded. It contains apoptotic and necrotic target cells, and the serum in the medium can effectively neutralize trypsin activity. Residual trypsin will severely interfere with subsequent apoptosis detection if not terminated properly.
d. Transfer the cell suspension into a suitable centrifuge tube, centrifuge at 1000 rpm for 5 minutes at room temperature, then aspirate and discard the supernatant.
e. Collect all cells obtained from step a and step d.
f. Resuspend the cells with PBS and perform cell counting.
g. Harvest 5×10⁴ to 1×10⁵ cells, centrifuge at 1000 rpm for 5 minutes at room temperature, then aspirate and discard the supernatant.
h. Wash the cells with 100 μL of Component A, centrifuge at 1000 rpm for 5 minutes at room temperature, then aspirate and discard the supernatant.
2. Cell Staining
(1) Resuspend the cells with 100 μL of Component A.
(2) Add 5 μL of Component B (Annexin V-R-PE), 1 μL of Component C (DRAQ7) and 1 μL of Component D (Hoechst 33342).
(3) Incubate at room temperature for 15–30 min in the dark (the incubation time can be optimized according to the preliminary experimental results).
3. Washing and Resuspension
(1) After incubation, centrifuge at 1000 rpm at room temperature for 5 min to collect cells.
(2) Aspirate and discard the supernatant, add 100 μL of Component A, and resuspend gently for washing.
(3) Centrifuge again, and completely aspirate off the supernatant.
(4) Add 500 μL of Component C to gently resuspend the cells to prepare samples for flow cytometry detection.
4. On-board Detection
(1) Important: Perform flow cytometry detection within 1 hour after staining to ensure stable fluorescence signals.
(2) Analyze samples using a flow cytometer with preset detection channels.
(3) First adjust the voltage with blank control (unstained cells) to locate the cell population at the lower-left corner of the coordinate system.
(4) Then detect the negative control and positive control to confirm the validity of the experimental system.
(5) Finally test the experimental samples, and record and analyze the mean fluorescence intensity of cells.
Annexin V-R-PE for early apoptotic signal detection: Ex/Em: 488/578 nm;
DRAQ7 for necrotic cell signal detection: Ex/Em: 599~644/678~697 nm;
Hoechst 33342 for viable cell signal detection: Ex/Em: 350/461 nm.
III. Result Interpretation
Qualitative Analysis (Microscopy)
Figure 1 All-in-One Apoptotic, Necrotic and Healthy Cell Comprehensive Status Enhanced Detection Kit (Annexin V-R-PE, Draq 7, Hoechst 33342) fluorescence microscopic imaging
The negative control group of K562 cells was cultured under normal conditions as the control; the positive control group was treated with the apoptosis inducer CCCP. After 16 hours, cells were stained with the kit and observed under a fluorescence microscope. To clearly distinguish fluorescent signals, PE fluorescence was pseudo-colored green and DRAQ7 fluorescence pseudo-colored red in the microscopic signal acquisition system.
(1) Negative control: Normal K562 cells, composite imaging after co-staining with Annexin V-R-PE, DRAQ7 and Hoechst 33342;
(2) Positive control: Apoptosis-induced K562 cells, composite imaging after co-staining with Annexin V-R-PE, DRAQ7 and Hoechst 33342.
Result Interpretation
(1) Early apoptotic cells: Significant externalization of cell membrane phosphatidylserine (PS) with strong red fluorescent signal of Annexin V-R-PE;
(2) Late apoptotic cells with damaged membrane: Positive for Annexin V-R-PE red fluorescence, DRAQ7 far-red fluorescence and Hoechst 33342 blue fluorescence;
(3) Necrotic cells: Only positive for DRAQ7 far-red fluorescent signal;
(4) Viable cells: Double negative for red/far-red fluorescence, with uniform blue nuclear fluorescence staining.
After apoptosis induction, the level of PS externalization increased significantly compared with the negative control, indicating obvious PS eversion on the cell membrane. The proportion of late apoptotic cells was markedly elevated (significantly increased intensity of far-red/red/blue fluorescence and number of triple-positive cells), suggesting that the inducer promoted cell apoptosis and further led to cell death.
Note: Actual test results may vary due to differences in testing instruments, operating conditions, cell lines and other factors. This example is for reference only.
FAQ
1. Q: Can fixed cells be used before or after staining?
A: Only live unfixed cells are applicable. Cell fixation causes artificial death and massive false positive apoptotic/necrotic signals.
2. Q: Why retain original culture medium for adherent cell flow assay?
A: Apoptotic/necrotic cells detach into medium and must be included for accurate ratio calculation. Serum in medium neutralizes trypsin to avoid Annexin V degradation and false negative results.
3. Q: Why total cell count and late apoptotic/necrotic proportion are lower than expected?
A: Damaged apoptotic/necrotic cells are easily lost in supernatant during centrifugation. Reserve small volume of supernatant to preserve loose cell pellets.
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