Passaging adherent cells
When a vessel fills up, detach the cells and move a fraction into a new vessel. The split ratio sets when you will split next, and trypsin exposure affects cell health.
Key points
- Split at 70–90% confluence
- Neutralise trypsin as soon as cells detach
- Always record the passage number
Materials
- Complete medium (warmed to 37 °C)
- PBS without Ca²⁺/Mg²⁺
- Trypsin-EDTA (0.05–0.25%)
- New flasks/dishes, centrifuge tubes
- Hemocytometer or automated counter
- Biosafety cabinet
Steps
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Split ratio
What it isWhat fraction moves to the new vessel. Higher ratios take longer to refill.
Lower ratioLow (e.g. 1:2): fills quickly, frequent splitting.
Higher ratioHigh (≥1:10): too sparse, some cells grow slowly or fail to attach.
Typical choiceDepends on the line — the ratio that reaches 80% in 2–4 days.
Trypsin time · concentration
What it isEnzyme treatment that detaches cells.
ShorterToo short: clumps remain and you select only easily detached cells.
LongerToo long: surface proteins are damaged; viability and attachment drop.
Typical choiceNeutralise as soon as cells detach (usually 2–5 min).
Confluence at splitting
What it isFraction of the surface covered by cells.
Lower · lessBelow 50%: few cells and slow recovery.
Higher · more100% for long: contact inhibition, differentiation and death increase; cells clump and detach poorly.
Typical choice70–90%, during log-phase growth.
Passage number
What it isHow many times the culture has been split.
HigherHigh: genetic and phenotypic drift can change drug response and expression.
Typical choiceFreeze low-passage stocks and experiment within a fixed range; report it in papers.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Cells do not detach | Over-confluent, residual serum | Rinse with PBS, slightly longer trypsin, scraper as last resort |
| Poor attachment after splitting | Over-trypsinisation, too sparse | Shorter trypsin, lower split ratio |
| Medium turns cloudy and yellow | Bacterial or fungal contamination | Discard; disinfect cabinet and incubator |
| Growth slows down | High passage, mycoplasma | Thaw a new stock, test for mycoplasma |
⚠ Safety Work in a biosafety cabinet of the appropriate level and treat human-derived cells as potentially infectious.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
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