View source for DEC2008-thawing-oral-cells

==THAWING OF NORMAL HUMAN ORAL CELLS==
# Turn on the water bath at 37 oC.
# Thaw the cryovial at 37oC quickly in the waterbath until the cell suspension becomes molten.
# Resuspend the cells in 10ml of their routine medium in a 15ml centrifuge tube.
# Centrifuge the cell suspension at 1200 rpm for 5 min.
# Resuspend the cells in a T25 cell culture flasks in their routine culture medium (5ml for T25 flask, 10ml for a T75 flask).
# Incubate in a humidified chamber with 5% CO2, at 370C.
# Change 2/3 of the medium the second day.

==SUBCULTURE OF NORMAL HUMAN ORAL FIBROBLASTS==
# Dilute 1:1 in PBS trypsin-EDTA 1x in a 15ml centrifuge tube (use 1ml trypsin 1x for each T25 and 2ml trypsin 1x for each T75 flasks) and place the tubes with trypsin in the water bath at 37ºC; warm it for 5min. If you use or TrypleX (this stays at room temperature) you do not need to warm it or dilute it.
# Discharge culture medium from the culture flask (at 80-90% confluence).
# Gently wash the cell culture with 2m/6ml RT PBS without Ca2+/Mg2+ (2ml for T25, 6ml for T75) for 1 min.
# Discharge PBS from the culture flask.
# Pipette the warm trypsin/Tryple X into the flask.
# Incubate it for 5-7 min at 37ºC.
# Check after 3 min whether the cells have detached after vigorous shaking of the flask.
# Add DMEM medium with 10% FBS 1:1 vol. into the flask (to neutralise the trypsin); mix it with the cell suspension in trypsin, and wash gently the bottom of the flask.
# Transfer cell suspension in a 15ml centrifuge tube.
# Centrifuge the cell suspension at 1200 rpm for 5 min.
# Resuspend the cells in new cell culture flasks (splitting ratio 1:4 or 1.6), in prewarmed DMEM routine culture medium (5ml for T25 flask and 10 ml for T75 flask).
# Incubate in a humidified chamber with 5% CO2, at 370C.
# Change 2/3 of the medium at each third day.

==SUBCULTURE OF NORMAL HUMAN ORAL KERATINOCYTES==
# Pipette trypsin-EDTA 1x in a 15ml centrifuge tube (use 1ml trypsin 1x for each T25 and 2ml trypsin 1x for each T75 flasks) and place the tubes with trypsin in the water bath at 37ºC; warm it for 5min.
If you use or TrypleX (this stays at room temperature) you do not need to warm it or dilute it.
# Put the tube with trypsin in the water bath at 37ºC; warm it for 5min.
# Discharge culture medium from the culture flask (at aprox. 80% confluence).
# Pipette the warm trypsin/TrypleX into the flask.
# Incubate it for 5 min at 37ºC.
# Check after 3 min whether the cells have detached after vigorous shaking of the flask.
# Add DMEM medium with 10% FBS 1:1 vol. (or only 1 vol. FBS) into the flask (to neutralise the trypsin); mix it with the cell suspension in trypsin, and wash gently the bottom of the flask.
# Transfer cell suspension in a 15ml centrifuge tube.
# Centrifuge the cell suspension at 1200 rpm for 5 min.
# Resuspend the cells in new cell culture flasks (splitting ratio 1:4 or 1:6), in prewarmed KSFM routine culture medium (5ml for T25 flask and 10 ml for T75 flask).
# Incubate in a humidified chamber with 5% CO2, at 370C.
# Change 2/3 of the medium at each third day.

==SUBCULTURE OF ORAL CANCER CELL LINES==
# Pipette trypsin-EDTA 1x in a 15ml centrifuge tube (use 1ml trypsin 1x for each T25 and 2ml trypsin 1x for each T75 flasks but sometimes you will need trypsin 10x to detach the cells) and place the tubes with trypsin in the water bath at 37ºC; warm it for 5min.
# If you use or TrypleX (this stays at room temperature) you do not need to warm it or dilute it.
# Put the tube with trypsin in the water bath at 37ºC; warm it for 5min.
# Discharge culture medium from the culture flask (at aprox. 80% confluence).
# Gently wash the cell culture with 2m/6ml RT PBS without Ca2+/Mg2+ (2ml for T25, 6ml for T75) for 1 min.
# Discharge PBS from the culture flask.
# Pipette the warm trypsin/TrypleX into the flask.
# Incubate it for 5 min at 37ºC.
# Check after 3 min whether the cells have detached after vigorous shaking of the flask.
# Add DMEM medium with 10% FBS 1:1 vol. (or only 1 vol. FBS) into the flask (to neutralise the trypsin); mix it with the cell suspension in trypsin, and wash gently the bottom of the flask.
# Transfer cell suspension in a 15ml centrifuge tube.
# Centrifuge the cell suspension at 1200 rpm for 5 min.
# Resuspend the cells in new cell culture flasks (you can go even up to a splitting ratio of 1:10), in prewarmed KSFM routine culture medium (5ml for T25 flask and 10 ml for T75 flask).
# Incubate in a humidified chamber with 5% CO2, at 370C.
# Change 2/3 of the medium at each third day.
# Freezing cells in liquid nitrogen
# Prepare 10% DMSO freezing medium by adding 1ml DMSO to 9ml FBS (5 ml DMSO to 45ml FBS) .
# Prepare the cryovials and label them with the name of the cells, passage no, approx no of cells frozen in each vial, your name and date.
# Trypsinize the cells.
# Centrifuge the cell suspension at 1200 rpm for 5 min.
# Resuspend the pellet in freezing medium at approx. 1 mil cells per ml.
# Aliquot in cryovials.
# Freeze them using the rapid method (follow the 2 step method and times indicated by the table that you will find in the freezing room) or just place the vials in liquid nitrogen.
# Write down in the catalog where the vials were placed in the tank.

[[Category:Protocols]]