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Co- culture experiment


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#1 Mohamed samir

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Posted 11 March 2013 - 09:01 AM

Dear ALl,

Alittle bit stricky for me to ask question like this.
Iam going to perform an experiment on a co- culture consists of both human A549 pneumocyte II cells and murine macrophage J774 cell line to test the effect of miRNAs on influenza virus replication .
I have no protocol for this and iam confused really whether I will be able to blend two cell line from different host. However, the media in both cells are differ , is this will pe appropriate medium for both cells ?
Should I used human macrophage cell line to be compatable with the A549 cell as it is from human ?
Dpepnding on which criteria, will our biculture succeed ?

Thanks

#2 Mohamed samir

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Posted 11 March 2013 - 09:20 AM

I can summarize the question
Can I culture two differenrt cell line ( for example, A549 and J774 ) at the time both of them was cultured in different medium ?
Can I culture two differenrt cell line ( for example, A549 and J774 ) that isolated from two different animal host or human ?

#3 bob1

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Posted 11 March 2013 - 11:59 AM

You can certainly culture two different lines in the same medium at the same time. This is why about 1/3 of the world's cell lines are HeLa...

You will have to wean one of the lines (probably best to do the A549, they are very robust) onto the other medium. You can do this by slowly changing the medium from old to new over several passages. For instance start with a ratio of (old:new) 9:1, then next passage change it to 7:3, etc, until it is fully the new medium. You have to be aware of the cells' morphology and growth rates while doing this so as to ensure that you are still working with cells that behave in the manner you would expect them to! This is very important if you are looking at cellular markers as changes in behaviour indicate that the cells have "evolved" and are no longer the cell line you think they are!

#4 Mohamed samir

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Posted 11 March 2013 - 04:49 PM

So, really this is not my question Iam asking if I can culture two cell line that was under single culture grown in two different medium, for example the A549 cell was noramylly grown in DMEM while Mcrophage J774 cell was grown in PRMI medium and now i want to culture both cell line together. Is this will be ok, which medium i will use to both culture ?

The second question, now i have A549 cell from human lung and the J774 is macrophage from mice. Is this ok if i culture them knowning that they are from different host

What do u mean by old and new , do you mean the old is the medium of A549 and the new is that of J774 ?

Thanks

#5 bob1

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Posted 12 March 2013 - 04:18 PM

OK, I thought that you wanted to grow them both in the same flask... So, yes, you can grow different cells in the same medium separately, there are many cell lines that use DMEM and many others that use RPMI, and yet more that use other different media.

I don't quite get your second question - people have been growing mouse cells and human cells in the same incubators (separate flasks) for well over 40 years.

Old is the medium you are weaning them off, new is the medium you are trying to change them onto.

#6 Mohamed samir

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Posted 13 March 2013 - 04:08 PM

ANother question please, If i will grow A549 and THP-1 cell in the same flask using two different medium. Do you know what is exact ration should be between both cells in terms of cell number. Is the different composition of the medium will affect the growth of both cell
note: i will grow both cell type in the same Flask.

Thanks and sorry for inconvenience

#7 rhombus

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Posted 14 March 2013 - 08:53 AM

Dear Mohamed,

I have grown J774 murine macrophages for nearly 30 years and have grown them in both DMEM and RPMI. They grow just as well in both media's and the choice of which one we use is dependent upon the experimental conditions that are important in that experiment. Population doubling times are similar, as is the induction of an enzyme we look at (inducible Nitric oxide synthase-iNOS)

The best way of co-culturing your cells for experiments is to do them in cell culture inserts i.e.

You grow the A549 in the 6 well plate and then add your cell culture insert and grow the J744 on your insert. The inserts normally have a pore size og 0.2-0.45uM so will not allow any of the J774 to go through the insert into the well of the 6 well plate BUT DOES ALLOW THE CONDITIONED MEDIA FORM THE 6 WELL PLATE TO THE INSERT.

Hope this helps

Kindest regards

Uncle Rhombus




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