📋 Workflow
Full workflow, products add-to-cart directly1
Cell preparation
1 hPrepare single-cell suspension from blood or spleen, lyse RBCs or separate by density gradient
💡 Tip Enrich rare subsets first
⚠️ Caution Viability affects phenotype reliability
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2
Surface staining
1 hAdd fluorochrome-conjugated antibody panel, incubate at 4°C in dark and wash
💡 Tip Use FMO controls for calibration
⚠️ Caution Protect from light to prevent quenching
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3
Intracellular staining (optional)
1 hFor cytokines/transcription factors, permeabilize then stain intracellular antibodies
💡 Tip Maintain permeabilized environment after step
⚠️ Caution Surface markers distort after permeabilization
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4
Acquisition and analysis
2 hAcquire on instrument, correct compensation with FMO/single-stains, gate to quantify immune subsets
💡 Tip Consistent gating ensures comparability
⚠️ Caution Watch spectral overlap in high-parameter panels
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Wrong compensation misclassifies subsets
- ② Surface markers distort after permeabilization
- ③ Debris/doublets disturb analysis
- ④ Poor panel design causes severe overlap
- ⑤ Missing FMO controls hamper positivity
❓ FAQ
+What are FMO controls?
Fluorescence-minus-one controls keep all dyes except test channel to set positive gates.
+How to analyze T/B/NK subsets?
Gate lymphocyte subsets by a CD3/CD19/CD56 marker panel.
+Can immune cells be sorted?
Yes, FACS-Sort physically separates subsets for culture or functional studies.