I am working on H9C2 cells and when I seed them with density of 5×105 cell/well in 24-well plate I see normal cells under microscope but in the day of apoptosis assay by annexin/PI and after the incubation with treatment, the cells die and flow cytometry always show cells number very low and double population appears too like another cells present .what is the problem please ?and how can fixe this?
Hello, Rofaida Messalem .
I think some small experiments can be added to find the cause of cell apoptosis.
1. Set up control groups:
Negative control (untreated cells): confirm the basal apoptosis rate (should be < 5%).
Positive control (such as 1 μM Staurosporine treatment for 4 hours): verify the apoptosis induction effect.
2. Confirm that the cells have indeed undergone apoptosis. Use trypan blue staining before and after treatment to distinguish apoptosis (intact cell membrane) from necrosis (damaged cell membrane). If a large number of cells float, it may be that the treatment conditions are too strong or the culture conditions are not suitable.
3. The flow parameters may be problematic:
Use single-stained samples (Annexin V + or PI+) to adjust compensation to avoid false positives caused by fluorescence crosstalk.
Other suggestions:
1. The seeding density can be adjusted. Inoculate 1×10⁵, 2×10⁵, and 3×10⁵ cells/well respectively, and compare the cell survival rate and apoptosis rate after treatment.
2. The treatment conditions need to be adjusted. When inducing with H₂O₂, you can set a concentration gradient (100, 200, 300 μM) and a time gradient (1, 2, 4 hours).
3. Add some measures to protect cell viability. After digestion, add culture medium containing 10% FBS to terminate trypsin activity and reduce mechanical damage.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
Rofaida Messalem We do quite a few apoptosis assays in our lab, and we've found that the Annexin V/PI stain can be a bit tricky to optimize. Since your cells appear healthy under the microscope and you're only seeing issues after staining, it’s possible that the staining procedure itself may need adjustment.
One thing to watch for: if you're resuspending your cells in PBS during PI staining, be sure to wash thoroughly with FACS buffer without calcium before switching to the Annexin V binding buffer, which does contain calcium. Residual phosphate from PBS can react with calcium to form calcium phosphate, which can be toxic and may lead to cell death—even in your untreated controls.
If you're seeing a high percentage of dead cells in your negative control, this could be the cause. For more details, you're welcome to refer to the methods section of this paper, which outlines the protocol we've used successfully in our lab:
When I seed the cells in a 12-well plate, they appear healthy after 24 hours, but after 48 hours, they begin to detach, die, and float in the media regardless of whether treatment is added. Sidney T. Sithole Lucian Miles
Rofaida Messalem , I'm glad to receive from you again.
Troubleshooting suggestions:
- Check the inoculation density, medium replacement frequency and composition, and plate surface treatment.
- Observe cell morphology changes, subculture in time, and avoid over-fusion. [Control the confluence of cells to 70-80% and subculture them promptly.]
- Check culture conditions (temperature, CO₂, contamination). [Ensure that the culture medium is fresh and replaced regularly, maintaining a pH of 7.0-7.6, 37°C, 5% CO₂ culture environment]
- Replace with high-quality FBS or try coating. [You can try to use coatings (such as gelatin, collagen) to improve adhesion.]
MCE (MedChemExpress) star product purchase address:
https://www.medchemexpress.com/search.html?q=gelatin&ft=&fa=&fp=&fsp=&ftag=&fsc=&fcc=&fol=&type=biochemical-assay-reagents
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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