Smolke:Protocols/Adherent cell care and feeding: Difference between revisions
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*Changing media - I aspirate out the media and add more media using a Pipet-Aid. I find that if I add the media too quickly the force of the liquid will dislodge the cells. This is undesirable. | *Changing media - I aspirate out the media and add more media using a Pipet-Aid. I find that if I add the media too quickly the force of the liquid will dislodge the cells. This is undesirable. | ||
*Subculturing - I aspirate out the media and add an amount of trypsin equal to about 1/2 the volume of the dish. As with changing media, I add slowly enough to avoid dislodging the cells. I then aspirate out the trypsin. This step removes the cells that adhere to the dish weakly or not at all. I add more trypsin, this time an amount equal to about 1/3 the volume of the dish. Dislodging the cells is no longer a concern. I return the dish to the incubator for about 5 minutes or until I can see the cells floating in the liquid rather than attached to the dish. I add an amount of media equal to about 2/3 the volume of the dish and pipet almost the whole volume up and down with the Pipet-Aid. This step breaks up any remaining clumps of cells and disperses them into a homogeneous suspension. | *Subculturing - I aspirate out the media and add an amount of trypsin equal to about 1/2 the volume of the dish. As with changing media, I add slowly enough to avoid dislodging the cells. I then aspirate out the trypsin. This step removes the cells that adhere to the dish weakly or not at all. I add more trypsin, this time an amount equal to about 1/3 the volume of the dish. Dislodging the cells is no longer a concern. I return the dish to the incubator for about 5 minutes or until I can see the cells floating in the liquid rather than attached to the dish. I add an amount of media equal to about 2/3 the volume of the dish and pipet almost the whole volume up and down with the Pipet-Aid. This step breaks up any remaining clumps of cells and disperses them into a homogeneous suspension. | ||
**If I am simply maintaining the cell line I | **If I am simply maintaining the cell line I may not count the cells. I add 40-100μL of the trypsinized cell mixture to a new dish of media. This method has generally worked for me. However, if the resulting cell density is too low (10% or lower) the cells will take a long time to repopulate the plate (up to a week) and even when they do they grow in dense colonies rather than evenly throughout the plate. This is undesirable. | ||
**If I am preparing for a transfection I will count the cells at this point, which allows me to calculate the concentration of the trypsinized cell mixture. After pipetting up and down with the Pipet-Aid again (the cells settle out of suspension in the time it takes to count using the hemocytometer) I dilute the cell mixture with media in a new Falcon tube to reach the concentration I want for the transfection, then aliquot the resulting mixture into the wells of a multi-well plate. | **If I am preparing for a transfection I will count the cells at this point, which allows me to calculate the concentration of the trypsinized cell mixture. After pipetting up and down with the Pipet-Aid again (the cells settle out of suspension in the time it takes to count using the hemocytometer) I dilute the cell mixture with media in a new Falcon tube to reach the concentration I want for the transfection, then aliquot the resulting mixture into the wells of a multi-well plate. | ||
Latest revision as of 17:36, 11 July 2012
Describe the procedures you use when working with adherent cells such as HEK. After everyone has posted contribution we will combine them to create a standard protocol.
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