I'm going to use microarray technology (Affymetrics) to study gene expression of 2 different cell types in coculture model . Does anyone know of a way to separate two cells or identify them to study their gene expression? Do you think using 2 different (spices to isolate the cells e.g mice vs rats) is a good idea to overcome this limitation?
If there are two different cell types then search for their characteristic cell membrane proteins like CD molecules (and if the cell types are different then one or two CD molecules must be their to capture), identify those proteins and you can get fluorophore tagged antibodies against those CD molecules from any vendor, then label the cells with those antibodies and sort the cell populations by FACS or cell sorter. Keep in mind that the two anti-CD antibodies must be tagged with different fluorophore. By this method, you will get both of your cells sorted and in pure population and you can use those cells for any downstream application you want.
I think the method of Dr. Kumar is a good choice by using FACS sorting. However, for your co-culture experiment, I suggest that a stable GFP transfection would be made on one of your target cell, and the other not. After the co-culture, cell sorting for GFP positive cells will get GFP-labeling cells in pure population and you can perform the microarray separately.
An alternative is to stain one cell type green and the other red. Check Invitrogen, they have several types of stain you can use. They work well and make cell sorting easy.
Good luck
Yes, as suggested by Hao, sorting for GFP expressing cells are easy but GFP expression may be sometimes problematic for cells' normal behaviour as its a foreign protein. And in co-culture experiments you have to take care of that as the only effect you want on your one cell line should be from another cell line. Therefore, I suggested the above mentioned method.
Instead of going for a microarray, why don't you seek if you can do a dual RNAseq. This is a very promising new technology, when you want to check RNA expression of two organisms in the same system. This is a deep sequencing approach, that means the number of reads for a gene is directly proportional to the RNA transcript copy. This is easy but may cost you higher ( although I think it would same as a Microarray experiment). Instead of spending your time separating cells, do this and this will also increase the impact of your work to be published in higher ranked journals.
Cheers,
Sudip
Thank you guys it is really helpful. I like the GFP idea. But as Sudip said it is a time consuming. Actually I don't know what the type of information can we got from RNA sequencing?.. Can you tell me more about it?
Rania
It all depends on what you want to do. When you say co-culture, I think you have either two different types of cells together physically or a cell type infected with a pathogen. In all these cases when you want to check the overall gene expression, you have to check the whole transcriptome for changes. In case of a dual RNA deep sequencing, you can check the overall changes in transcriptome of two organism, species thriving together. You do not need to separate the two entities. If you want further information, researchers at our University are doing this regularly.
http://www.nature.com/nrmicro/journal/v10/n9/full/nrmicro2852.html
You can also use FACS to separate the two types of cells by using a appropriate marker system. Harvest two cell types and then do a Microarray or a RNA deep sequencing to observe changes in gene expression.
http://bloodjournal.hematologylibrary.org/content/118/14/e101.short
Selection with antibodies via magnetic bead separation could be another option. But you need to consider that one of the two cells you work with will be tagged with antibody.
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