📋 Workflow
Full workflow, products add-to-cart directly1
Cell preparation and treatment
VariesDetach adherent cells and resuspend, or count suspension cells; seed in 6-well plates and treat per protocol
💡 Tip Pilot test treatment concentration and time first
⚠️ Caution Avoid over-digestion damaging cell membrane
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2
Collect and wash cells
30 minCollect supernatant and adherent cells, wash twice with PBS, resuspend in binding buffer
💡 Tip Include floating cells from supernatant
⚠️ Caution Use ice-cold PBS to reduce handling damage
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3
Dual staining
30 minAdd Annexin V-FITC and PI, incubate 15 min at RT in dark
💡 Tip Include single-stain and unstained controls for compensation
⚠️ Caution Run promptly; PI background rises over time
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4
Flow cytometry and analysis
1 hRun on flow cytometer, analyze four quadrants: live/early/late apoptosis/necrosis
💡 Tip Collect at least 10k events per tube
⚠️ Caution Keep consistent gating for comparability
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⚠️ 5 Common Beginner Mistakes
- ① Over-trypsinization causes false-positive apoptosis
- ② Missing supernatant cells loses late-apoptotic population
- ③ PI background grows with incubation causing false positives
- ④ Missing single-stain/unstained controls skews compensation
- ⑤ Inconsistent gating makes batches incomparable
❓ FAQ
+How to distinguish live/early/late apoptosis and necrosis?
AV−/PI− live, AV+/PI− early apoptosis, AV+/PI+ late apoptosis, AV−/PI+ necrosis.
+Why many PI-single-positive cells?
Often from mechanical damage, over-digestion or late collection causing secondary necrosis.
+Can fixed cells be used?
Fixation disrupts membrane and Annexin V binding; live cells are more reliable.