I am having a little difficulty with the basics of isolating cd133 cells from peripheral blood. My goal is to end up with 5 milliliters(or more) of cd133 cells. I am used to separating cells using a easysep magnet from stemcelltech, but the only kit I see available for cd133 cells is the microbead kit from miltenyibiotec. See here
https://www.miltenyibiotec.com/en/Products-and-Services/MACS-Cell-Separation/Cell-separation-reagents/Hematopoietic-stem-cells/CD133-MicroBead-Kit-human.aspx
and
https://www.miltenyibiotec.com/en/Products-and-Services/MACS-Cell-Separation/Cell-separation-reagents/Hematopoietic-stem-cells/Diamond-CD133-Isolation-Kit-human.aspx
My questions are
1.) Has anyone used these products before?
2.)I would just like someone to explain what the product is exactly. Does it work the same way as the cd34 isolation kit from stemcelltech?
3.) Is there another kit or way of isolation cd133 cells that you know of?
4.)HOW TO CULTURE THEM! lol I would just like to know of the appropriate mediums used for culture of cd133.
5.)How long would I have to culture them to end up with at least 5 milliliters of cells?
I would really just like instructions on how to use the kits I was referring to in the links above. It's my first time using them and am a bit scared to mess it up. So if anyone could be so kind as to give some brief step by stem instructions on how to use them to isolate cd133 cells.
I used to work for Miltenyi so I can give some insight into the CD133 kit, which works on a similar principle to kit you mention from SCT. However, because of the differences between the two technologies there are differences in the methods used. SCT Easysep uses a magnet which surrounds the sample, attracting cells to the edges of the tube, Miltenyi use a column based system which you place into a magnet and then load the cells. Therefore the Miltenyi system can get away with using smaller particles because of the increase in magnetism generated by the column, the SCT kit uses larger particles because overall the magnetism generated by the Easysep is less. Therefore the two technologies are not interchangeable and you will need a Miltentyi system to use the CD133 kit.
An alternative method to isolate the CD133 cells would be to isolate the MNC fraction by centrifugation, perform a magnetic sort using the CD34 Easysep kit and then FACS sort this fraction for CD133.
I can't too add too much detail about culture of these cells however could you clarify; by 5 milliliters do you mean 5 million cells?
I hope this is useful to you.
Csn you sort the cells? If they are CD133 PBMCs, they could probably be cultured on fibronectin coated plates + EBM. See article by Suuronen, 2006.
http://cardiovascres.oxfordjournals.org/content/70/1/126.full.pdf+html
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