Yes. Passage number is most often considered to be the equivalent of how many times the cells have been stripped of their surface protein by enzyme digestion. Often when working with primary cells, the cells that outgrow the initial plated tissue will be called P0. Then the cells are harvested by trypsin and the replated cells are P1.
The other time it is critical to consider passage number is during freezing. Everyone in the whole lab should work to the same principle. Some labs take trypsinised P0 cells and freeze them as P0. Then when they are replated the new flask is now labelled P1. Other labs label the frozen tubes as P1 and plate them on the new plastic as P1.
and I would not re-use the same flask after trypsin: 1-risk of contamination, 2-risk that the plastic characteristics change 3-risk of having cells partially digested still attached. Point 1 and 3 you can avoid if you re-sterilize the flask, but keep in mind point 2...you would never know....
i accept Fulvio valuable reason.. i have one more doubts.. we were maintained A549, MCF7, INS cell lines if i should maintain upto 50 passage or more.
1). Normally How many passage cells are used MTT or some other assay?
2). If we are maintained still 100 passage is good r not?
3). A549 and MCF7 are cancer cell line, passage is an important parameter for research studies mean why? Explain Briefly?
Yes. For adherent cells, passage number increases each time they are trypsinized and reseeded. For suspension cells, if you follow a recommended spit ratio for maintenance of commercially available cells, that is a good estimate of incremental passage.
Note, for many adherent lines, if trypsinized cells are reseened into the same dish or flask, their morphology is often different. I always seed into a new dish/flask.
For adherent cells: every time you detach cells and let them attach again... Simply that's one passage. No matter if they remain in the same flask. In other words, one passage represents the starting point for a new morpho-rearrangement of the culture; this is what has to be kept in mind when passing more and more times cultured cell lines.
For cells in suspension: every time you divide the cell number giving cells more volume of medium, you are renewing the stimulus for division/differentiation (you're changing the concentrations of inducing factors and signals released by cells).
The number of passages possible will depend on the type of cells being grown, and the conditions of culture. Primary cells will often have a limited number of doublings before becoming senescent. Canerous cell lines contain mutations to block senescense/apoptosis and these will grow for a very long time. A good example is HeLa cells, and the story of HeLa cells (google Henrietta Lacks) is worthwhile reading up on from a science history/ethics point of view.
However, it is important to remember that these immortalized cells lines may evolve over time with multiple passages. It would be a good idea to keep frozen stocks from early passages, which can be thawed and cultured if either the morphology or growth rate of your line changes over time.
Deifnately. Why are you reusing the same flask? Would advise using a new flask each time. Its also important to not go too High in terms of passage number. Would also regularly freeze down batches of cells so for exp purposes can use cells of similar passage number.
Deifnately. Why are you reusing the same flask? Would advise using a new flask each time. Its also important to not go too High in terms of passage number. Would also regularly freeze down batches of cells so for exp purposes can use cells of similar passage number.
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