📋 Workflow
Full workflow, products add-to-cart directly1
Collect and fix cells
1 hCollect and wash cells, add ice-cold 70% ethanol dropwise, fix overnight at 4°C
💡 Tip Add ethanol dropwise with vortex to prevent clumping
⚠️ Caution Thorough fixation gives even staining
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2
Pre-stain treatment
30 minWash off ethanol with PBS, add RNase to remove RNA interference
💡 Tip Adequate RNase avoids RNA tailing
⚠️ Caution Wash thoroughly to remove ethanol
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3
PI staining
30 minAdd PI staining solution, incubate 30 min at RT in dark
💡 Tip Protect from light to avoid quenching
⚠️ Caution Excess PI risks precipitation
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4
Flow and fitting
1 hRun on flow cytometer, use software to exclude doublets and fit G1/S/G2 peaks
💡 Tip Slow sample rate lowers CV
⚠️ Caution Doublet exclusion is key to accuracy
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Too-fast ethanol fixation causes clumping
- ② Insufficient RNase causes RNA tailing disturbing fits
- ③ Undue doublet exclusion inflates G2/S
- ④ High PI background enlarges CV
- ⑤ Cell death before fixation skews cycle distribution
❓ FAQ
+Distinguish S from G2/M?
By DNA content; S is continuous, G2/M peak is 2× G1.
+What is a good CV?
CV <5% is good; larger indicates uneven staining or poor fixation.
+Can apoptosis be measured simultaneously?
Fixation disrupts Annexin V; use separate batch or other markers.