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HEK293T cell culture & passaging

Mammalian cell culture

Routine adherent culture of HEK293T cells in 10 cm dishes. Same protocol works for HEK293, HeLa, and other adherent lines with minor adjustments.

Complete medium (DMEM-10)

• DMEM, high glucose (4.5 g/L), with GlutaMAX or L-glutamine — 445 mL
• Heat-inactivated FBS — 50 mL (10% final)
• Penicillin/Streptomycin (100×) — 5 mL (1× final, 100 U/mL P, 100 µg/mL S)

Filter through a 0.22 µm bottle-top filter if mixing yourself. Store at 4 °C, use within 1 month.

Routine passaging (10 cm dish, 1:5–1:10 split)

1. Pre-warm DMEM-10, 0.05% trypsin-EDTA, and DPBS at 37 °C in the bead bath.
2. Aspirate medium from the dish (cells should be 70–90% confluent).
3. Wash gently with 5 mL DPBS. Aspirate.
4. Add 1 mL trypsin-EDTA. Tilt to coat. Incubate 37 °C, 2–3 min, until cells round up (HEK293T detach quickly — don't over-trypsinise).
5. Tap the dish sharply to release cells. Add 4 mL DMEM-10 to neutralise trypsin.
6. Pipette up and down 4–5 times against the dish bottom to dissociate clumps. Don't make foam.
7. Transfer to a 15 mL conical. Optional: count cells (see cell counting).
8. Pellet 200 × g, 5 min. Aspirate.
9. Resuspend in 5–10 mL fresh DMEM-10. Plate the desired fraction into a new 10 cm dish containing 9 mL fresh medium.
10. Rock the dish in cross pattern (left-right, then forward-back) to distribute cells evenly. Avoid swirling — clumps cells in the centre.
11. Return to 37 °C, 5% CO₂.

Splitting ratios

Handling notes

• HEK293T are loosely adherent — be gentle with media changes or you'll lift them off.
• Passage every 2–3 days to maintain log-phase growth. Don't let them go fully confluent.
• Discard cells past passage ~30 (drift accumulates) and revive a fresh vial. Track passage number on the dish.
• Test for mycoplasma every ~2 months. See mycoplasma testing.
• Always pre-warm media — cold shock impairs growth.