Zhang A, Pan S, Zhang Y, et al. Carbon-gold hybrid nanoprobes for real-time imaging, photothermal/photodynamic and nanozyme oxidative therapy. Theranostics. 2019;9(12):3443-3458. Published 2019 May 24.doi:10.7150/thno.33266(IF:8.063)
When reading literature, researchers—especially those working in cell biology and pharmacology—frequently encounter beautiful flow cytometry apoptosis figures. Among various detection methods, Annexin V-based flow cytometry assays are among the most widely used for assessing cell apoptosis.
However, for many, flow cytometry remains a confusing technique. Adding apoptosis analysis to the mix often results in strange, unexpected plots. You might wonder: How do I troubleshoot these odd results or optimize my protocol?
Worry no more—we’ve compiled a comprehensive list of common problems in Annexin V apoptosis assays!
Operation Section
1. What is the principle of Annexin V-FITC/PI apoptosis detection?
In early apoptosis, phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. Annexin V labeled with FITC (green fluorescence) binds specifically to the externalized PS, indicating early apoptotic cells. Propidium iodide (PI), a red-fluorescent DNA-binding dye, penetrates only cells with compromised membranes, such as late apoptotic or necrotic cells. PI can be excited by 488, 532, or 546 nm lasers.
2. Are Annexin V/PI apoptosis kits species-specific?
No. Annexin V binds to PS, which is conserved across species. Therefore, the kits are not species-dependent.
3. Does using trypsin with EDTA affect the apoptosis detection?
Yes. Annexin V binding is Ca²⁺-dependent. EDTA chelates Ca²⁺, thereby interfering with Annexin V-PS interaction and compromising assay results.
4. If my cells express GFP, which apoptosis kit should I use?
Avoid FITC-labeled Annexin V (same emission as GFP). Use kits labeled with PE, APC, or Alexa Fluor 647 to minimize spectral overlap.
5. Why should platelets be removed from blood samples?
Platelets contain PS, which can bind Annexin V and produce misleading results. Removing them avoids interference.
6. What precautions should be taken when handling Annexin V staining reagents?
Annexin V dyes are light-sensitive. Perform staining and incubation steps in the dark, and analyze the samples within 1 hour of staining for best results.
Result Section
1. How to perform fluorescence compensation correctly?
Theoretically, X-mean and Y-mean should be equal(As shown in the figure), but practically, the goal is to prevent fluorescence spillover.
For example, FITC-only stained cells should not appear in the PI-positive quadrant.
Recommended controls:
Unstained control: For adjusting FSC/SSC and voltage settings to define the population.
Single-stain controls: Induce apoptosis and stain separately with Annexin V-FITC and PI.
Double-stained sample: Acquire data using parameters optimized from controls.
2. What causes false positives in control groups?
Poor compensation causing fluorescence overlap. Re-adjust using proper controls.
Overconfluent or starved cells may undergo spontaneous apoptosis.
Over-trypsinization or mechanical damage can disrupt membrane integrity.
Interfering fluorescence from drugs or cell autofluorescence.
Delayed flow cytometry after staining or prolonged drug treatment.
3. Why are there no positive signals in the treated group?
Drug concentration or treatment duration may be insufficient.
Apoptotic cells in the supernatant are missed. Always include the supernatant.
Operational errors: missing dye addition or washing away dye after staining.
Kit degradation or improper storage. Use a positive control to verify kit functionality.
4. What if only nuclear signal is positive and Annexin V is negative?
Poor cell health. Use healthy, log-phase cells.
Excessive pipetting or harsh handling. Be gentle to avoid mechanical damage.
5. What if only Annexin V is positive but nuclear dye is not?
Nuclear dye (PI or 7-AAD) may have been omitted. Repeat staining with proper dye.
Cells may be in early apoptosis only. Adjust drug treatment conditions and observe under a microscope.
6. What if the cell populations are not clearly separated?
Autofluorescence of cells may interfere with signal. Choose a kit with a non-overlapping fluorophore.
Poor cell condition can cause nonspecific PS exposure. Use gentle dissociation enzymes like Accutase.
Summary:
Before the experiment, determine whether your cells have autofluorescence and select a compatible apoptosis detection kit. Use gentle, EDTA-free dissociation enzymes such as Accutase. Avoid omitting dyes, and do not wash cells after staining. During analysis, ensure that voltages and compensation are correctly set, and always use positive controls to verify kit performance. For treatment groups, design proper concentration and time gradients to determine optimal experimental conditions.
This way, your apoptosis plots can be just as publication-worthy as those in top-tier journals!
Product Overview
|
Product Name |
Specification |
Cat No. |
|
20 T/50 T/100 T |
40302ES20/50/60 |
|
|
20 T/50 T/100 T |
40303ES20/50/60 |
|
|
20 T/50 T/100 T |
40304ES20/50/60 |
|
|
20 T/50 T/100 T |
40305ES20/50/60 |
|
|
Annexin V-PE/7-AAD Apoptosis Detection Kit |
20 T/50 T/100 T |
40310ES20/50/60 |