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
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Grow cells until 80 % confluent, cells should be healthy and split no more 2-3 days before the freeze down.
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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.
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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)
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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.
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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)
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Aliquot 0.3 ml of resuspended cells to each plate.
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Check with the Fisher Micromaster Microsope to see if the cells resuspended well to single cells.
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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