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Tissue Culture

Protocols for thawing, splitting, and maintaining adherent cell lines.

Cell Thawing Procedure

Set Up

Allow ~30 minutes before starting.

Sign up for the sterile hood (~1 hr). Warm DMEM + 10% FBS + 1:100 Penn/Strep to 37 °C. Sterilize hood: turn on UV 30 min before and spray with 70% ethanol.

Materials

  • DMEM + 10% Fetal Bovine Serum (FBS) + 1% Penn/Strep (Media)
  • 5 mL and 10 mL pipet tips
  • 25 cm² T-flasks (T-25)
  • 15 mL conical tube
  • 70% Ethanol

Procedure

  1. Obtain cell vials from the liquid nitrogen tank in tissue culture room #286D (with label fields for cell details).
  2. Allow cells to thaw, then add 1 mL of warm media.
  3. Label a new T-25 flask: cell line, date, passage #, and initials.
  4. Use a 15 mL conical to add 4 mL of media + 1 mL of cells = 5 mL total.
  5. Spin at 1.25 rpm (×1000) for 5 min.
  6. Aspirate old media.
  7. Add 4 mL of media to the cell pellet and resuspend (pipet up and down), then add cells to a T-25 flask (spread liquid evenly).
  8. Loosen the caps and place into a 37 °C / 5% CO₂ incubator.
  9. Clean up:
    • Add 10% bleach to waste flask.
    • Aspirate.
    • Throw away used materials in the biohazardous waste trash.
    • Spray down hood with 70% EtOH and wipe.
    • Leave hood on for the next person. Otherwise, turn off blower and close hood completely.

Adherent Cell Splitting Procedure

Set Up

Allow ~30 minutes before starting.

Sign up for the sterile hood (duration ~1 hr). Warm DMEM + 10% FBS + 1:100 Penn/Strep and 1× PBS to 37 °C. Sterilize hood: turn on UV 30 min before and spray with 70% ethanol.

Materials

  • DMEM + 10% Fetal Bovine Serum (FBS) + 1% Penn/Strep (Media)
  • 1 mL, 5 mL, and 10 mL pipet tips
  • 2× 25 cm² T-flasks (T-25)
  • Sterile 1× PBS
  • Trypsin
  • 70% Ethanol

Procedure

  1. Obtain cell flask from the incubator in tissue culture room #286D.
  2. Check for 80–100% confluency (no gaps or overlapping) using a 10× zoom light microscope.
  3. Aspirate old media.

  4. Wash with sterile 1× PBS — 5 mL (T-25) or 10 mL (T-75).

    • Spread PBS evenly.
    • Aspirate.

    Note

    Stickier cells (e.g., A431s) need 2× washes.

  5. Add 0.5 mL (T-25) or 1 mL (T-75) Trypsin (protease — releases cells from flask wall).

    • Spread Trypsin evenly.
    • Incubate at 37 °C for 5–10 min.

    Note

    Stickier cells need 1 mL Trypsin.

  6. Prep T-25 or T-75 flask.

    • Label: cell line name, passage #, date, initials.
  7. Remove cells from incubator.
    • Tap T-flask to get cells to the bottom.
    • Add media accordingly for the correct dilution (e.g., 1:2 — add 2 mL, take 1 mL; or 1:5 — add 5 mL, take only 1 mL for each T-25) with a total of 5 mL (T-25) or 10 mL (T-75) with 10% FBS + DMEM.
    • Pipet up/down.
    • Pipet media along the wall and break up cell clumps.
  8. Add to 2× newly labeled T-flasks.

  9. Incubate at 37 °C and 5% CO₂ for 2–3 days.

    Tip

    Cells double in approximately 24 hours.

  10. Clean up:

    • Add 10% bleach to waste flask.
    • Aspirate.
    • Throw away used materials in the biohazardous waste trash.
    • Spray down hood with 70% EtOH and wipe.
    • Leave hood on for the next person. Otherwise, turn off blower and close hood completely.