This is a very open ended question, so I will try and give definitions for my answers. Firstly in the case of purifying growing the cells over the time period of the experiment one does not have to worry to much as this is really a primary culture and one does not need to consider the continued proliferation of the cells. Katrin makes a really good suggestion, and If I were doing the experiment I would adopt that strategy. However a cell line is a different proposition. Most cells in primary culture will proliferate until the reach their Hayflick limit, which is a point at which they will no longer grow. This will generally be not much more 60 cell divisions, depending on the initial source of the cells etc. This is in part determined by the length of the telomeres in your initial isolation. If your goal is to generate a stable and immortal cell line you can leave these cells in culture, even if they do not grow until they reach a point when they actually start growing, however this can take a very very long time. Now if your Hayflick limit is a long way off from your starting point you could create a pool of primary cells which you can expand and then use over a set number of passages to perform your experiments. However your cells will more than likely end up loosing some of their primary characteristics as they may well have changed or accumulated mutatations or changes that make them different as they are in effect being selected for their increased growth potential. If you want to actually generate a stable and immortal cell line then you can actually transfect/infect with a telomerase expressing vector, and the ATCC has used this to generate cells with extended proliferative capacity. Another approach is to use a temperature sensitive or conditional SV40 Large T antigen expression system which allows you to make these cells immortal.

If it were me doing the experiment and I had access to a readily available source of primary tissue I would just purify the cells a Katrin has said and do my experiments in a primary culture. This has the benefit that the cells will most likely behave in a manner that is closer to their natural behaviour in vivo, and the different number of primary cultures should give you a wide spread of phenotype from a number of different donar individuals or animals. Secondly, the time it takes to generate and characterise a stable and immortal cell line can be quite long and all the transfections/infections and selection might generate a series of heterogeneous cell lines with very little resemblance to the primary tissue.

Good luck

Try isolating the cells first and then culturing in different media

u can use either cell lines of your interest from the database such as ATCC (American Type Culture Collection), or use primary cells isolated from different tissue.

thank you very much for your answer and for your concerd Dr Himanshi and Dr Bashir,

How I can isolate a certain types of cells? for example how i can isolate Macrophages and construct a cell line from these cells?

you can use e.g. MACS to isolate specific cell types via magnetically labeling of cells.

Thank you very much dr katrin for your answer

This is a very open ended question, so I will try and give definitions for my answers. Firstly in the case of purifying growing the cells over the time period of the experiment one does not have to worry to much as this is really a primary culture and one does not need to consider the continued proliferation of the cells. Katrin makes a really good suggestion, and If I were doing the experiment I would adopt that strategy. However a cell line is a different proposition. Most cells in primary culture will proliferate until the reach their Hayflick limit, which is a point at which they will no longer grow. This will generally be not much more 60 cell divisions, depending on the initial source of the cells etc. This is in part determined by the length of the telomeres in your initial isolation. If your goal is to generate a stable and immortal cell line you can leave these cells in culture, even if they do not grow until they reach a point when they actually start growing, however this can take a very very long time. Now if your Hayflick limit is a long way off from your starting point you could create a pool of primary cells which you can expand and then use over a set number of passages to perform your experiments. However your cells will more than likely end up loosing some of their primary characteristics as they may well have changed or accumulated mutatations or changes that make them different as they are in effect being selected for their increased growth potential. If you want to actually generate a stable and immortal cell line then you can actually transfect/infect with a telomerase expressing vector, and the ATCC has used this to generate cells with extended proliferative capacity. Another approach is to use a temperature sensitive or conditional SV40 Large T antigen expression system which allows you to make these cells immortal.

If it were me doing the experiment and I had access to a readily available source of primary tissue I would just purify the cells a Katrin has said and do my experiments in a primary culture. This has the benefit that the cells will most likely behave in a manner that is closer to their natural behaviour in vivo, and the different number of primary cultures should give you a wide spread of phenotype from a number of different donar individuals or animals. Secondly, the time it takes to generate and characterise a stable and immortal cell line can be quite long and all the transfections/infections and selection might generate a series of heterogeneous cell lines with very little resemblance to the primary tissue.

Good luck

thank you very much Dr Andrew for your good informations