📋 Protocol
Full flow at a glance, add products directly1
Preparation (Aseptic Setup)
30 minUV-sterilize the biosafety cabinet, disinfect surfaces, prepare sterile consumables and reagents.
💡 Tip Spray everything entering the hood with 75% ethanol.
⚠️ Caution Work without blocking the laminar airflow.
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2
Thaw Cells
20 minRemove vial from liquid nitrogen, thaw rapidly at 37°C, centrifuge to remove freezing medium, seed into a dish.
💡 Tip Thaw quickly in a 37°C water bath (within 1–2 min) to reduce ice-crystal damage.
⚠️ Caution Freezing medium contains DMSO — centrifuge it out after thawing.
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3
Media Change
15 minAspirate old medium, rinse with PBS, add fresh complete medium.
💡 Tip Pre-warm medium to 37°C before use to avoid cold shock.
⚠️ Caution Rinse gently with PBS to avoid detaching adherent cells.
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4
Passaging (Trypsinization)
30 minAt 80–90% confluency, detach with trypsin, neutralize, and split at a ratio.
💡 Tip Keep trypsinization short; stop as soon as cells round up under the microscope.
⚠️ Caution Suspension cells need no trypsin — centrifuge and split directly.
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5
Cell Counting
15 minStain with trypan blue, count viable cells with a hemocytometer for consistent seeding density.
💡 Tip Trypan blue excludes live cells; blue cells are dead.
⚠️ Caution Mix thoroughly before counting to avoid clumps.
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6
Cryopreserve Cells
30 minResuspend in DMSO-containing freezing medium, cool to -80°C gradually, then transfer to liquid nitrogen.
💡 Tip Freeze slowly, thaw quickly — use a controlled-rate freezing container.
⚠️ Caution DMSO final concentration 5–10%, prepare fresh.
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⚠️ 5 Common Beginner Mistakes (Pitfall Guide)
- ① Contamination: mycoplasma is most common — test regularly and stay aseptic.
- ② Over-trypsinization: damages membranes, lowers viability — stop when cells round up.
- ③ Passaging too late: over-confluency causes contact inhibition/differentiation — passage at 80–90%.
- ④ Wrong freeze/thaw: reversing slow-freeze/fast-thaw kills many cells.
- ⑤ Serum batch variation: different FBS lots affect cells — lock to one lot when possible.
❓ FAQ (Troubleshooting)
+Cells growing slowly or poorly?
Check: (1) serum quality/lot, (2) medium expiry, (3) mycoplasma contamination, (4) split ratio.
+How to tell if it's mycoplasma?
Slow growth, morphology change but medium stays clear. Confirm with a PCR detection kit.
+Adherent cells suddenly detaching?
Likely over-confluency, nutrient depletion, pH change, or contamination. Change medium, passage, and check for contamination.