Haynes:SplittingCells

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* 1x Phosphate-buffered saline (PBS)
* 1x Phosphate-buffered saline (PBS)
* Trypsin-EDTA buffer/ media
* Trypsin-EDTA buffer/ media
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* Complete cell culture medium
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* Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)
==Procedure==
==Procedure==
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# Add PBS to the cells. Lay the flask flat and wash by gently tilting the flask back and forth.<br>Note: For a T-75 flask, use 5 mL PBS. For a T-25, use 2 mL PBS.
# Add PBS to the cells. Lay the flask flat and wash by gently tilting the flask back and forth.<br>Note: For a T-75 flask, use 5 mL PBS. For a T-25, use 2 mL PBS.
# Aspirate off the PBS.
# Aspirate off the PBS.
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# Add Trypsin-EDTA into the culture flask. Lay the flask flat and coat the cells completely with the Trypsin-EDTA. Let the flask sit in the hood at room temperature for 5 minutes.
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# Add Trypsin-EDTA into the culture flask. Lay the flask flat and coat the cells completely with the Trypsin-EDTA. Let the flask sit in the hood at room temperature for 5 minutes.<br>Note: For a T-75 flask, use 2 mL PBS. For a T-25, use 0.5 mL PBS.
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7.) After incubation, examine the cells under a microscope.  Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask/dish.
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# After incubation, examine the cells under a microscope.  Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask/dish.
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8.) If the cells are not fully detached, place the flask back into the incubator.  Some cells may require some mechanical agitation (including “rapping” the flask or “scraping” the culture surface), BUT THIS IS NOT PREFERRED.
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# If the cells are not fully detached, place the flask back into the 37 °C incubator.  Some cells may require some mechanical agitation (including “tapping” the flask).
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9.) Once the cells have detached, add serum-containing medium to the flask in an amount approximately 2-3X that of the trypsin (i.e., 10-15ml of medium for a T-75 culture flask).  Trypsin will start to act on the excess serum proteins instead of harming the cells.
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# Once the cells have detached, add '''serum'''-containing medium (e.g., 10% FBS) to the cells to deactivate the trypsin.<br>Note: For a T-75 flask, use 8 mL medium. For a T-25, use 3.5 mL medium.
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Note: The medium MUST contain serum in order to act to inhibit the trypsin. Serum-free media can only be used IF a trypsin inhibitor is used.
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# Get a new flask, label it with the cell line name, your initials, and the date. Include the passage number (e.g., PS-1 if you are splitting the cells for the first time, PS-2 for the second time, etc.).
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10.) Collect the harvested cells and pipet into an appropriately sized centrifuge tube.
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# If you are splitting 1:10, add 9 mL medium to the new flask. In the old flask, gently resuspend the cells by pipetting up and down about 5 times. Transfer 1 mL of cells into the 8 mL of medium in the new flask. Lay the flask flat and tilt back and forth to spread the cells evenly.<br>Note: For less robust cells,split 1:5 by adding 2 mL cells to 8 mL fresh medium, or 1:4 by adding 2.5 mL cells to 7.5 mL medium.
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11.) Centrifuge cells for approximately 5 minutes at 200xg (800-1100 rpm, depending on the centrifuge).
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# Place the new flask into the 37 °C incubator. The cells should attach in a couple of hours.
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12.) During centrifugation, label new culture flasks.  Label flasks with cell type, your initials, the new passage number (passage number increases with every split), and today’s date.
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13.) NOTE: Certain cell types are only viable up to a certain passage number, such as primary cells.  Make sure to check this before splitting beyond the appropriate passage number!
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14.) Following centrifugation, aspirate the media above the cell pellet and resuspend the cells in a logical volume (5-10 ml).
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15.) If necessary, count cells via hemacytometer or coulter counter.
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16.) Resuspend your pelleted cells in an appropriate volume of growth medium and dispense into sterile flasks or onto your experimental surfaces.
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Revision as of 17:33, 13 May 2013

<- Back to Protocols

Adherent Cells

Materials

  • 1x Phosphate-buffered saline (PBS)
  • Trypsin-EDTA buffer/ media
  • Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)

Procedure

  1. Pre-warm all reagents to 37°C in the bead bath.
  2. After reagents are warmed, spray bottles down with ethanol and prepare the hood as for routine work.
  3. Aspirate old culture media from the cell culture vessel.
  4. Add PBS to the cells. Lay the flask flat and wash by gently tilting the flask back and forth.
    Note: For a T-75 flask, use 5 mL PBS. For a T-25, use 2 mL PBS.
  5. Aspirate off the PBS.
  6. Add Trypsin-EDTA into the culture flask. Lay the flask flat and coat the cells completely with the Trypsin-EDTA. Let the flask sit in the hood at room temperature for 5 minutes.
    Note: For a T-75 flask, use 2 mL PBS. For a T-25, use 0.5 mL PBS.
  7. After incubation, examine the cells under a microscope. Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask/dish.
  8. If the cells are not fully detached, place the flask back into the 37 °C incubator. Some cells may require some mechanical agitation (including “tapping” the flask).
  9. Once the cells have detached, add serum-containing medium (e.g., 10% FBS) to the cells to deactivate the trypsin.
    Note: For a T-75 flask, use 8 mL medium. For a T-25, use 3.5 mL medium.
  10. Get a new flask, label it with the cell line name, your initials, and the date. Include the passage number (e.g., PS-1 if you are splitting the cells for the first time, PS-2 for the second time, etc.).
  11. If you are splitting 1:10, add 9 mL medium to the new flask. In the old flask, gently resuspend the cells by pipetting up and down about 5 times. Transfer 1 mL of cells into the 8 mL of medium in the new flask. Lay the flask flat and tilt back and forth to spread the cells evenly.
    Note: For less robust cells,split 1:5 by adding 2 mL cells to 8 mL fresh medium, or 1:4 by adding 2.5 mL cells to 7.5 mL medium.
  12. Place the new flask into the 37 °C incubator. The cells should attach in a couple of hours.
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