Tissue Culture Protocol

2015-05-31

azim58 - Tissue Culture Protocol

Tissue Culture Protocol
passaging cells
passage cells
passage tissue cells

An example basic protocol for a T175 flask: wash w/PBS (about 10 mL), 2.5 mL trypsin (wait a few min for cells to lift), add media with sera (about 8 mL), centrifuge, aspirate, resuspend to desired volume, count cells if needed

First decide dimensions/volumes. See
tissue culture volumes and dimensions page
or
tissue culture volumes and dimensions from Danielle page

Wash cells with ___mL of 1X PBS

o Release PBS on the side of the flask the cells are not on
o cap the flask, and gently move the flask so that the PBS washes
the side with the cells

Aspirate away the 1X PBS
Detach cells with ___mL of 0.05% Trypsin

o Release Trypsin on the side of the flask the cells are not on
o cap the flask, and gently move the flask so that the trypsin goes
over the cells

Incubate for about 2 minutes
Hit the flask so the cells will release

o may want to check status of cells under the microscope at this
point

Neutralize the trypsin by adding ___mL of media

o Release media on the side of the flask the cells are not on

cap the flask, and gently move the flask so that all the trypsin is

neutralized

If passing, first decide what ratio you'd like to do it. Remove all

but 1 mL of media/cells and add media to the necessary corresponding
volume.

Put date on front of the flask and what passage number it is. Return

to 37C incubator, 5% CO2.

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Protocol from the Current Protocols in Cell Biology (2007):
Tissue culture protocol_Current protocols in cell bio.pdf

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Starting Tissue Culture Cells

Cell Lines