I am going to attempt using PDGFRB tagged PE antibodies to attempt to isolate these cells from mice kidneys and to culture these cells ( if successful). Would appreciate some advise on this if you have some experience in this.
We have done this with FACS. You can get a decent number of cells form an injured, or fibrotic kidney, after IR or UUO for example. But the problem is getting enough cells from a control or uninjured kidney, since these have very few Pdgfr positive fibroblasts.
I have some experience with MACS but not with PDGFRB. My advice would be to make sure your cells will stain for IF with the same antibody you are using for the sorting. If the antibody doesn't bind well on IF, you won't get adequate binding for associating your cells with the beads. I believe there are Rabbit, Mouse and Streptavidin conjugated heads available so this might broaden your options.
I use the MS columns and find any more than 25 million cells takes a long time to pass (the PI says 50 million but I add another column if sorting more than 25 million - I'm also sorting kidney cells). Keep everything on ice and work fast and smart. Finally the Miltenyi Biotech protocol says to plunge the plunger into the column after it is removed from the magnet and MACS buffer added. I use chilled media instead (whatever I'm planning on culturing them in) which obviates the need to spin them down again prior to culture. Also, I apply gentle pressure only with the plunger and it is clear that the cells come out in the first few drops so no need to be aggressive with flushing them out in my opinion.
Ultimately the quality of the MACS will be determined by the quality of the dissociation and you will need single cells. I use Accutase and a 20um vacuum cell strainer to maximise separation and yield.
Best of luck!
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