Tissue Culture

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Cell Thawing Procedure

Date:

Set Up

~30min before starting
Sign up for sterile hood (~1hr)
** Warm DMEM +10% FBS +1:100 penn/strep to 37°C
Sterilize hood: turn on UV 30min before and spray with 70% Ethanol**

Materials:

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

Procedure:

1. Obtain cell vials from liquid nitrogen tank from tissue culture room #286D
Stick label:
Cell details:

Stick label:
Cell details:

Stick label:
Cell details:

2. Allow cells to thaw then add 1mL of warm media

3. Label new flask T-25: Cell line, date, passage #, and initials

4. Use 15mL conicals to add 4 mL of Media + 1mL of cells = 5mL total

5. Spin at 1.25 rpm (x1000) for 5min

6. Aspirate old media

7. Add 4mL of Media to cell pellet and resuspend (pipet up and down), and 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:

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

Adherent Cell Splitting Procedure

Date:

Set Up

~30min before starting
Sign up for sterile hood (Duration ~1hr)
** Warm DMEM +10% FBS +1:100 penn/strep and 1x PBS to 37°C
Sterilize hood: turn on UV 30min before and spray with 70% Ethanol**

Materials:

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

Procedure:

1. Obtain cell vials from liquid nitrogen tank from tissue culture room #286D

Cell details:

Cell details:

Cell details:

  1. Check for 80-100% confluency (no gaps or overlapping) using a 10x zoom light microscope
  2. Aspirate old media
  3. Wash with sterile 1x PBS, 5mL (T-25) or 10mL (T-75).
  • Spread PBS evenly
  • Aspirate

Note: stickier cells need 2x washes (i.e. A431’s)

4. Add 0.5mL (T-25) or 1mL (T-75) Trypsin (protease, release cells from flask wall)
Note: stickier cells need 1mL Trypsin

  • Spread Trypsin evenly
  • Incubate at 37 ˚C for 5-10min

5. Prep T-25 or T-75 flask

  • Label: Cell line name, Passage #, Date, Initials

6. Remove cells from incubator

  • Tap T-flask to get cells to the bottom
  • Add media accordingly for the correct dilution (ex: 1:2 add 2mL take 1mL, or 1:5mL add 5mL only take 1mL for each T-25) with a total of 5mL (T-25) or 10mL (T-75) with 10% FBS + DMEM
  • Pipet up/down
  • Pipet media along the wall and break up cell clumps

7. Add to 2x newly labeled T-flask

8. Incubate at 37 ˚C & 5% CO2 for 2-3 days.
Note: Cells double in approximately 24 hours

9. Clean up:

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