We used C57BL/6 mice at the age of 8–12 weeks where the development and maturation of RPE had been completed 59 . The mice were euthanized by CO2 inhalation and cervical dislocation. The eyes were then enucleated and bisected along the equator. The eyecups were sectioned in Ames' solution (Sigma‐Aldrich) with 10 mM HEPES and pH adjusted to 7.4, and the retina was gently removed leaving the RPE firmly attached to the eyecup. To isolate the RPE cells for patch‐clamp recordings, the eyecup was incubated at 37°C in 5% CO2 either in TrypLE Select for 15 minutes or in a solution containing (in mM) 135 TeaCl, 5 KCl, 10 HEPES, 3 EDTA‐KOH, 10 glucose, and 25 U/ml activated papain (Sigma‐Aldrich) for 30 minutes. After this, the eyecups were washed in the HEPES buffered Ames' solution supplemented with 1% bovine serum albumin (BSA; Sigma‐Aldrich). The RPE was collected by gentle trituration, stored at 37°C in 5% CO2 in the RPE culture medium and measured within 6 hours.
Ames solution
Manufactured by Merck Group
Sourced in Germany, United States
Ames solution is a microbiological culture medium used for the Ames test, a widely used method for detecting mutagenic compounds. It consists of a nutrient-rich growth medium formulated to support the growth of specific bacterial strains. The core function of the Ames solution is to provide a controlled environment for conducting the Ames test, which is a standard assay for evaluating the mutagenic potential of chemical substances.
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Lab products found in correlation
Plasma cleaner, by Diener Electronic (1 mentions)
Forene 100, by Abbott (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Activated papain, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bx51wi, by Olympus (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
Nitrocellulose membrane disc, by Merck Group (1 mentions)
15 protocols using ames solution
1
Isolation and Culture of Mouse RPE Cells
2
Multiscale Retinal Electrophysiology
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Mice were dark-adapted overnight by placing their home cage in a light-shielded box fitted with an air pump for circulation. All dissection procedures the day of the experiment were carried out in complete darkness using infrared converters and cameras. Mice were decapitated, eyes enucleated, and placed into oxygenated room temperature Ames solution (Sigma, A1420) during retinal dissection and vitrectomy as described previously (Yao et al., 2018 (link)). An ~1 × 2 mm piece from dorsal retina was placed RGC side down on an MEA with either 512 electrodes spaced 60 µm apart, or 519 electrodes with 30 µm spacing (Field et al., 2010 (link); Frechette et al., 2005 (link); Ravi et al., 2018 (link)). Oxygenated Ames perfused the retina throughout the experiment at a rate of 6–8 mL/min, heated to 32°C.
3
Retinal Electrophysiology in Murine Models
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After Euthanasia by an overdose of isoflurane (Forene 100% [vol/vol]; Abbott GmbH, Wiesbaden, Germany) and decapitation, eyes of rd10 and C57BL/6J (wild type) mice of post natal week (PNW) 2, 4, 12, 20 as well as eyes of MNU-treated wild type mice were enucleated and transferred into oxygenated AMES solution (Sigma Aldrich, Germany). The eye was opened along the ora serrata, the lens and vitreous were removed. The retina was separated from the eyecup and was flattened with the photoreceptor side on a piece of nitrocellulose with a precut window. After removing of remaining fluid the retina was placed with the ganglion cell side down onto the 60 TiN electrodes of a planar 8x8 multi electrode array (MEA) with an electrode spacing of 200 μm and an electrode diameter of 30 μm (Multichannel Systems, Reutlingen, Germany). MEAs were pretreated with a plasma cleaner (Diener Electronic, Ebhausen, Germany) and coated with 0.25 mg/ml poly-D-lysine hydrobromide for about 2 h (Sigma Aldrich, Germany).
4
Retinal Electrophysiology in Rat Eye Cups
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Eyes were enucleated after decapitation of deeply anesthetized wild-type Long-Evans rats, in accordance with institutional guidelines for the care and use of animals. Immediately after enucleation, the anterior portion of the eye and vitreous were removed in room light, and the eye cup was placed in a bicarbonate-buffered Ames’ solution (Sigma-Aldrich, St. Louis, MO). Under infrared illumination, pieces of retina 3 to 5 mm in diameter were isolated from the sclera and placed ganglion cell side up on dialysis membrane in the perfusion chamber. The microwire bundle was lowered into the chamber until it made contact with the retina, holding it in place against the membrane. The preparation was perfused with Ames’ solution bubbled with 95% O2 and 5% CO2 and maintained at roughly 30°C and pH 7.4 using an in-line heater. Spontaneous recordings were performed under low, ambient light, and pulsed stimulation consisted of a white, full-field pulse delivered with a handheld flashlight lasting roughly 1 s, followed by roughly 4 s of darkness. The recorded data were filtered (band pass, 300 to 6000 Hz), and spike sorting was performed using Mountainsort as discussed in the “Data processing and analysis” section.
5
Retinal Tissue Isolation for Electrophysiology
6
Multielectrode Recordings from Rhesus Macaque Retina
7
Extracellular Recording of Rat Retinal Ganglion Cells
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Retinal tissue was obtained from adult (8 weeks old) male Long-Evans rat (Rattus norvegicus) and continuously perfused with Ames Solution (Sigma-Aldrich) and maintained at 32 °C. Ganglion cell spikes were recorded extracellularly from a multi-electrode array with 252 electrodes spaced 60 μm apart (custom fabrication by Innovative Micro Technologies, Santa Barbara, CA). Experiments were performed in accordance with institutional animal care standards. The microelectrode covered a total retinal area of ∼ 1 mm2. For the rat this corresponds to 16-17 degrees of visual angle [25 (link)]. The spike sorting was performed with an in-house method based on [22 (link)].
8
Murine Retinal Tissue Isolation
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All procedures were approved by the Institutional Animal Care and Use Committee at the University of Michigan. C57BL/6 mice aged 6–10 weeks were used in this study. Mice were anesthetized with an intraperitoneal (IP) injection of ketamine (100 mg/kg)/xylazine (10 mg/kg). Cornea, lens, and vitreous body were removed, and the retina was isolated from the pigment epithelium and cut into four pieces. Retina pieces were placed on filter article with ganglion cells layer up. The filter article with tissue was mounted in a chamber of an upright microscope (Olympus BX51WI) with 40× water immersion lenses. The chamber was continuously perfused with Ames solution (Sigma-Aldrich, St. Louis, MO USA; 4–6 ml/min) and equilibrated with 95% O2 5% CO2 at 37°C.
9
Retinal Experiments in Dark-Adapted Mice
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All retinal experiments were approved by the Duke University Animal Care and Use Committee. Adult mice (2–6 months, C57Bl/6J, Jackson Laboratories, 000664) of both sexes were used. Animals were kept on a 12-hour light/dark cycle with ad lib access to food and water. Prior to use, animals were dark-adapted overnight by placing the animal in a light-shielded box fitted with an air pump for circulation. All dissection procedures the day of the experiment were carried out in complete darkness using infrared converters and cameras. Mice were decapitated, eyes enucleated and placed into oxygenated room-temperature Ames solution (Sigma, A1420) during retinal dissection and vitrectomy as described previously [100 (link)].
10
Murine Eye Immunostaining Protocol
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C57BL/6 mice at the age of 8-12 weeks were used in this study for immunostaining. The mice were euthanized by CO2 inhalation and cervical dislocation, after which the eyes were enucleated and bisected along the equator. The eyecups were sectioned and the retina removed in Ames’ solution (Sigma-Aldrich) with 10 mM HEPES, pH adjusted to 7.4 with NaOH.
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