Smolke:Protocols/Adherent cell care and feeding

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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. Jay General comments: I feed or subculture my cells every 48-72 hours. I monitor the density of my cells using the light microscope and estimate the percentage of the dish surface the cells are covering. If the cells are at 60% or lower I will change the media; 70% or higher I will subculture. Whenever I aspirate I use the glass Pasteur pipet sheathed with a clean L1000 pipet tip. 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. If I am simply maintaining the cell line I do 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. Leo General comments: Changing media - Subculturing - Yvonne General comments: comments here refer specifically to HEK cells. Volumes 24-well plate: 0.5 ml/well 6-cm dish: 3 ml total 10-cm dish: 10 ml total I was taught to use these volumes by Steph/Chase. However, please note that they don't actually scale with the growth areas of the dishes (2 cm^2 for 24-well plate, 21 cm^2 for 6-cm dish, and 58 cm^2 for 10-cm dish). Therefore, plates/dishes seeded at the same density in terms of million cells/ml will not have the same confluency. General techniques Lift lid and tilt plate with one hand while aspirating/pipetting with the other The lid should cover the plate as much as possible, providing just enough opening to perform the required task The lid opening should face away from the hood opening (i.e., away from you and toward the back wall) Tilt the plate toward the back when aspirating Eject media gently against the side wall of the dish to avoid dislodging cells When aspirating, immerse the pipet tip in the liquid but don't touch the plate until the very end to avoid aspirating away cells As with all cell culturing, it is desirable to be as consistent as possible in protocol and timing. Therefore, I always count the cells when I subculture and seed at the same density each time (unless I have specific reasons to seed at a different density). I also feed the cells on a regular schedule. Changing media Aspirate away old media and add fresh media as described above; add antibiotics (for selection pressure) as necessary If seeding 6-cm dish at 0.02 million cells/ml (see below), feed cells 3 and 5 days after seeding; cells should be ready for subculturing on day 6 Subculturing Aspirate away old media Wash plate with trypsin (800 ul for 6-cm dish; 3 ml for 10-cm dish) and quickly aspirate away Add another volume of trypsin (same as above) and store in 37C incubator for 10 min Neutralize with media (1 ml for 6-cm dish; 3 ml for 10-cm dish Mix well by pipetting Sample 10 ul and place in 600-ul microfuge tube Dilute with 90 ul media (make sure it's well mixed) Add 12.5 ul diluted cells to 12.5 ul Trypan Blue Count cells with hemocytometer: count 4 quadrants and divide by 20 to get million cells/ml Seed new dish of the appropriate size at 0.02 million cells/ml Ryan General comments: Changing media - Subculturing - Kathy General comments: Changing media - Subculturing -