General Mammalian Cell Culture Protocol
Standard Operating Procedure (SOP) for adherent cell lines
Materials Required
- ✓ Complete growth medium (appropriate for cell line)
- ✓ Fetal bovine serum (FBS)
- ✓ Penicillin-Streptomycin (optional)
- ✓ PBS (without Ca²⁺/Mg²⁺)
- ✓ Trypsin-EDTA solution
- ✓ T25/T75 culture flasks or plates
- ✓ 37°C CO₂ incubator (5% CO₂)
- ✓ Centrifuge & Biosafety cabinet
1. Cell Thawing Protocol
1. Pre-warm complete culture medium to 37°C.
2. Remove cryovial from LN₂ → thaw in 37°C water bath (gentle agitation).
3. Thaw rapidly until small ice crystal remains (~1–2 min).
4. Disinfect vial with 70% ethanol before entering biosafety cabinet.
5. Transfer cells to centrifuge tube with 5–10 mL pre-warmed medium.
6. Centrifuge at 1000 rpm for 3–5 min.
7. Remove supernatant, resuspend pellet in fresh complete medium.
8. Transfer to culture vessel → incubate at 37°C, 5% CO₂.
9. Replace medium after 12–24 hours (removes DMSO & dead cells).
2. Routine Cell Culture
- Observe morphology & confluency daily under microscope.
- Replace culture medium every 2–3 days.
- Maintain at: 37°C / 5% CO₂ / 95% Humidity.
3. Cell Passaging Protocol (Adherent)
1. At 70–90% confluency → remove spent medium.
2. Wash gently with PBS (without Ca²⁺/Mg²⁺).
3. Add Trypsin-EDTA to cover cell layer.
4. Incubate at 37°C for 1–5 min (monitor detachment).
5. Neutralize trypsin with complete medium.
6. Transfer to centrifuge tube → spin at 1000 rpm, 3–5 min.
7. Resuspend in fresh medium → seed at desired split ratio.
Recommended split ratios:
➤ Fast-growing cells:
➤ Slow-growing cells:
➤ Fast-growing cells:
1:5 to 1:10➤ Slow-growing cells:
1:2 to 1:4
4. Cryopreservation Protocol
1. Harvest cells during logarithmic growth phase.
2. Centrifuge → resuspend in freezing medium:
90% FBS + 10% DMSO.3. Adjust density to 1–5 × 10⁶ cells/mL.
4. Aliquot 1 mL per cryovial.
5. Place in controlled-rate freezing container → -80°C overnight.
6. Transfer to liquid nitrogen for long-term storage.
5. Important Notes
✔ Always work under sterile conditions.
✔ Use authenticated & mycoplasma-free cell lines.
✔ Avoid repeated freeze-thaw cycles.
✔ Morphology/growth may vary between cell lines.
✔ Follow biosafety regulations.
Disclaimer: This protocol is a general guideline for mammalian cell culture. Specific cell lines may require optimized conditions, media, or handling procedures. Always refer to the data sheet for your cell line.