I do not know this protocol but you may want to try chelating bivalent cations (like Ca2+) with EDTA and using DNAse 1 to decrease clumping/aggregation of debris. Also, the more you vortex, the more ROS you'll get but also the more they may brake up (and the more cells will be liberated from the retina) leading to getting more debris and cells that can clump... so you may want to adjust that step.

Hi Peter,

Thanks for your suggestions. The application for these ROS is for phagocytosis assay on RPE cells cultured in vitro. Would you suggest using an other protocol for ROS isolation?

Julien-

I have done phagocytosis assays using pig ROS using the protocol described here for cow eyes. Pig works just as well, just double the number of eyes in the prep to account for their smaller size.

Molday, R.S., D. Hicks, and L. Molday. 1987. Peripherin. A rim-specific membrane protein of rod outer segment discs. Invest. Ophthalmol. Vis. Sci. 28:50–61.

I don't think it's a super-pure preparation but it's certainly sufficient for doing phagocytosis assays. I modeled my experiments after what Silvia Finnemann has done in her lab, and this is the procedure that she has referenced.

Hi Joseph,

Thanks for the reference. I just checked Silvia Finnemann protocol, and although it is more complicated that the one I used it may be worth trying it.

hi Julien, I think the protocol you are using is too crude. some sort of gradient purification of the ROS from other cell debris is needed. But of course it depends also on what you wish to use the ROS for. And, yes, it is a somewhat tedious procedure.

If you decide to modify the protocol you tried, one thing I suggest is to avoid harsh sheaing such as vortexing etc entirely. In my experience ROS can easily be aggregated or destroyed this way. There is no need for this either. In solution of whole retina, simply hand-shaking will make the ROS separate from the photoreceptor cell body.

There are alternatives to mechanical dissociation of photoreceptors from the retina, namely enzymatic treatments. Papers by David Hicks and colleagues describes this approach well, but other groups have had success with this method especially for maintaining photoreceptors in cell culture media for prolonged periods.

In addition, the purity of the dissociated photoreceptors can be greatly enhanced by density gradient centrifugation using a variety of media, including sucrose (standard), Ficoll (see Schnetkamp, 1981), and Percoll.

For pure ROS you can see the papers from P. Schnetkamp (around the 80's). But if you want to study phagocytosis, may be you dont need to have them very pure(see paapers from Hall, for that). For the prep you have, I would try : to disrupt tissue in Buffer and overlay the sample in sucrose-buffer and centrifuge, after that you can speed down the ROS.

Thanks a lot for all your answers and suggestions. In order to get a better idea of my ROS preparation purity I plan to run a SDS gel and see how big the rhodopsin band is.

I believe, as Silvia pointed out, that I have been a bit too harsh during the first steps of retina agitation. And will definitely try to use gradients the next times.

The protocol I used was described for use in phagocytosis assay, and looked simple enough for someone with no experience isolating ROS, which is why I decided to try it first.

Hi Julien, I hope you figured out the protocol for isolating ROS. Is is possible to get a copy of protocol ?

Thanks

Hi Julien,

I found this thread here from 6 years ago, I am trying to isolate porcine outer segments . Can you please send me the protocol that you used finally? I would appreciate your help.