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Mammalian Cell Culture Techniques Pdf Free

 
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MessagePosté le: Ven 7 Oct - 05:20 (2016)    Sujet du message: Mammalian Cell Culture Techniques Pdf Free Répondre en citant




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Read about our cookie policy. Then carry out one of the appropriate following procedures:Make sure flasks are labelled with the cell line, passage number, split ratio, date, operator initials and the vial number of the cells.Place flask(s) straight into 37C CO2 incubator. Tip flask gently a few times to rinse the cells and carefully pour/pipette the PBS back out into waste pot.This may be repeated another one or two times if necessary (some cell lines take a long time to trypsinize and these will need more washes to get rid of any residual FBS to help trypsinization)Using pipette, add enough trypsin EDTA to cover the cells at the bottom of the flask.e.g. Write down the details of the sub-culturing in the culture record log sheet. If you continue without changing your settings we'll assume youre happy. Note that most cells must not be split more than 1:10 as the seeding density will be too low for the cells to survive.As a general guide, from a confluent flask of cells:1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days.Attached cell line split ratios are done on volume of flask surface area:1 x 25cm2flaskSplit 1:3 or;3 x 25 cm2 flasks1 x 75 cm2 flaskSuspension cell line split ratios are done on volume of culture cell suspension:25ml cell suspension+75 ml fresh media in 5 seperate new flasks100 ml cell suspensionSplit 1:4 or;50 ml cell suspension+150 ml fresh media in 2 larger flasksIf cells are less then 70-80% confluent but you wish to subculture them on (eg Friday before the weekend) then they should be split at a lower split ratio in order to seed the cells at a high enough density to survive e.g.

There should be a separate log sheet for each vial of cells resuscitated and in use.7)Sub-culturing loosely attached cell lines requiring cell scraping for sub-cultureWhen ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100 ml of 10% sodium hypochlorite) taking care not to increase contamination risk with any drips.Replace this immediately by carefully pouring an equal volume of pre-warmed fresh culture media into the flask.Using cell scraper, gently scrape the cells off the bottom of the flask into the media. Some suggestions: Go back to the last page Go to the home page .. Some fast growing cells may require a high split ratio to make sure they do not overgrow. However, some cells, particularly primary cells, will require growth on special matrixes such as collagen to promote cell attachment, differentiation or cell growth.We recommend reviewing the relevant literature for further information on the cells you are culturing.The following is an example for endothelial and epithelial cells:For human cells, coat flasks with 1% gelatin. Culture media and supplements should be sterile. in 25 cm2 flask approx 1 ml75 cm2 flask approx 5 ml175 cm2 flask approx 10 mlRoll flask gently to ensure trypsin contact with all cells. Change media to get rid of any residual trypsin.9) Sub-culturing of suspension cell linesCheck guidelines for the cell line for recommended split ratio or sub-culturing cell densities.Take out required amount of cell suspension from the flask using pipette and place into new flask.e.g. ..

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