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Make sure no hand or clothes over the plates or tubes with cells, close the cap after each adding or withdrawal. 


Cell Growth and Split protocol

  1. Grow cells until 80 % confluent, cells should be healthy and split no more 2-3 days before the freeze down.

  2. Trypsonize Cells. Remove media from plate. For 10 cm plate, wash with 1 ml of PBS buffer. Tilt plate in both directions making sure the entire plate is covered.  Aspirate PBS.  Add another 1 ml of Trypsin. Tilt again to ensure full coverage of plate.  Incubate cells at 37 °C in CO2 until they release from plate.  Usually 5-10 minutes.

  3. Label 3 of 10 cm plates using the standard format given below. Add 10 ml DEME containing 10% FBS to each plate. (For 15 cm plates, add 25 ml to each plate)

  4. Examine plates with the Fisher Micromaster Microsope to ensure that all cells are unattached from the plastic and not over-trypsonize the cell (some cluster means good trypsonization, too many single cells means over). Add 1 ml DEME-10%FBS media from the new plate (not open too many times of stock DEME-10%FBS).  Pipitte media over tilted plate to wash down cells where they can then be collected into a 15 ml falcon tube.  Centrifuge cells for 1 min @ 1500 RPM.  

  5. Remove Media and Trypsin from tube, without disturbing cell pellet (tilt tube and aspirate media with Pasteur where tip is at least 1 inch away from cell pellet).  Add 1 ml DEME-10%FBS media from the new plate to the cell pellet and pipette vigorously to break up all cells. (use the 1 ml pipette to break up by withdraw and push only 100 µl each time pipetting to avoid the cell to touch the pipette)

  6. Aliquot 0.3 ml of resuspended cells to each plate.

  7. Check with the Fisher Micromaster Microsope to see if the cells resuspended well to single cells.

  8. Incubate cells at 37 °C in CO2 for 3 days and check growth progress.


Labeling Information

*all labeling should be done with a fine tip Sharpie marker

*Label plate with the date, cell line name and passage number 

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