I am just starting to culture MCF-10A. I read in ATCC that for subculturing MCF-10A we need soybean trypsin inhibitor and 0.05% trypsin-0.2% EDTA. Are these two exactly necessary for the subculture? If I replace 0.05% trypsin-0.2% EDTA with 0.25% trypsin-0.038% EDTA will that create problem?
I will be obliged if anybody could help me regarding this.
Thank You
Using **0.25% trypsin-0.038% EDTA** is feasible but requires protocol adjustments to avoid cell damage. **SBTI remains essential** for trypsin neutralization in serum-free conditions. Always validate with microscopic checks and pilot experiments.
- Rinse cells with PBS (Ca²⁺/Mg²⁺-free).
- Add 0.25% trypsin-0.038% EDTA (4 mL/T-75 flask).
- Incubate at 37°C for **2–3 minutes**.
- Neutralize with SBTI (180 µL per 4 mL trypsin)[3].
- Centrifuge and resuspend in fresh medium.
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