Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Summary

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 °C.

Abstract

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

Introduction

In the retina, vision is triggered by isomerization of photosensitive molecules called opsins, before being transformed into a signal that can be transmitted between neurons up to the visual areas in the brain. These molecules are embedded in stacks of membranous disks resembling pancakes that constitute the outer segment portions of photoreceptor cells (PRs). Being subjected to constant exposure to light and therefore considerable levels of oxidative stress, PRs continuously renew their outer segments to limit potential oxidative damage. Photoreceptor outer segments are in close contact with apical microvilli of the neighboring retinal pigment epithelial (RPE) cells.....

Protocol

This POS isolation experiment is time consuming and may require up to 12 hr to complete if POS are labeled before storage. The protocol has been adapted from a paper published by R.S. Molday and colleagues in 1987³¹ and modified by S.C. Finnemann and colleagues in 1997¹⁰.

Animals were handled according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Protocols were reviewed and approved by the Charles Darwin Ethics Committee from University Pierre and Marie Curie-Paris 06 and the Fordham University Institutional Animal Care and Use Co....

Results

The combination of the linear sucrose gradient and the ultracentrifugation allows the separation of the different components of the retinal suspension by density. Heavy larger retinal debris and RPE cells sink to or near the bottom of the gradient (Figure 2A). Lighter POS and lighter individual cells or cell debris from the retina migrate as separate bands to reach the top half of the gradient by the end of the centrifugation period. By keeping the eyes and the samples in the dark until after the centrif.......

Discussion

Three steps or conditions are crucial for an optimized purification: quality of gradient casting and delicate gradient tubes manipulation, keeping tissues chilled and in the dark until the collection step, strength of shaking of retinal homogenates to obtain proper POS isolation from the rest of the PR cell. If some issues arise in seeing the orange band properly, they are most likely due to one of the three reasons above (see also the second paragraph of the Results section). Some modification of certai.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Specific Material/Equipment
2 Chamber gradient maker			Gradient maker with 30 ml chambers
3 mm diameter silicone tubing			tubing for gradient casting
Small size magnetic stir bar			Stir bar fitting the gradient maker chamber
Red safelight lamp			Inactinic lamp for dissection in the dark
Ultra Clear 25 x 89 cm tubes	Beckman	344058	Ultracentrifugation tubes
PP Oak Ridge tubes	Nalgene	3119-0050	30 ml centrifugation tubes
Optima LE-80K	Beckman Coulter	365668	Ultracentrifuge
SW 32Ti swing rotor	Beckman Coulter	369694	Swing rotor for ultracentrifuge
Avanti J-26 XP	Beckman Coulter	393124	Centrifuge
JA-25.50 rotor	Beckman Coulter	363058	Rotor for Avanti J-26 XP centrifuge
FITC Isomer I	Life Technologies	F-1906	Fluorescent dye
Other Material/Equipment
Counting chamber (such as Neubauer or Malassez)
Dark ice buckets with lids
Scales
Magnetic stirrer and upholding pole
Refrigated microcentrifuge
37 °C water bath
-80 °C freezer
Consumables
Labcoat			Health and safety
Gloves
Sleeve protectors
Goggles
Absorbent pads
Biohazard trash bags and bins
Weck-Prep blades		Dissection	60 mm/2.25 inch wide razor blades
15 cm plastic dish
Sterile gauze sheets
15 and 50 ml tubes			Common consumables
Microtubes
Aluminum foil