I'm hoping to isolate RPE cells from mice eyes and perform PCR, however, contamination from the retina will interrupt my results. I'm wondering how people might have tried this in the past. I know complete removal of retinal contamination is very hard, but I'd at least like to minimize it.
Right now I just mechanically separate the retina from the RPE with forceps after enucleation. This does a decent job at getting most of the retina off the RPE, but there's still some cross contamination. I've tried using enzymes like dispase/hyaluronidase but it hasn't worked great for me. Any advice would be great! Thanks!
One way to isolate RPE cells from mouse eyes without retinal contamination is to use laser capture microdissection (LCM). LCM is a technique that uses a laser to cut out a specific cell type from a sample, allowing for the isolation of a specific cell type from a heterogeneous population. After the laser has been used to isolate the RPE cells, they can then be collected and further studied.
Hi Sonali, here are strategies you can try to improve the purity of your RPE cell population. Here are a few suggestions:
Enzymatic dissociation: Instead of solely relying on mechanical separation, you can try enzymatic dissociation methods to help separate the RPE cells from the retina more effectively. Enzymes like collagenase or trypsin can be used to break down the tissue and facilitate cell dissociation. Optimizing the concentration and duration of enzymatic treatment, as well as the buffer composition, may help improve the efficiency of cell separation.
Cell sorting: If your research facility has access to cell sorting technologies like fluorescence-activated cell sorting (FACS), you can use specific markers to isolate RPE cells directly. RPE cells express markers such as RPE65 or Bestrophin-1, which can be targeted with fluorescently labeled antibodies for sorting. This method can provide highly purified RPE cell populations.
- Badges
- Science method