Explants from E6.5 retinas were cultured in vitro on Millicell culture plate inserts according to the method described by Stoppini et al. [46 (link)] with some modifications [19 (link)]. Retinas were dissected out into cold Gey’s balanced salt solution (Sigma, St. Louis, MO) supplemented with 5 mg/mL glucose (Sigma) and 50 IU-μg/mL penicillin-streptomycin (Invitrogen, Paisley, United Kingdom). After removing the pigment epithelium, square explants (3 mm x 3 mm) were isolated from the central area of each retina and then placed on Millicell inserts (Millicell CM, 30 mm, pore size 0.4 μm, Millipore, Bedford, MA) vitreal surface down. Millicell inserts were placed in six-well plates containing 1 mL/well culture medium composed of 50% basal medium with Earle’s salts, 25% Hank’s balanced salt solution, 25% horse serum, 1 mM L-glutamine, 10 IU-μg/mL penicillin-streptomycin (all purchased from Invitrogen), and 5 mg/mL glucose. E6.5 retina explants were then incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 hours in vitro (E6.5+24hiv retina explants).
Gey s balanced salt solution
Manufactured by Merck Group
Sourced in United States
Gey's balanced salt solution is a laboratory reagent used to maintain the physiological environment for cells in vitro. It is a buffered saline solution that provides essential ions and nutrients to support cellular growth and function.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
D glucose, by Merck Group (3 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Millicell cm, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Hank s balanced salt solution, by Merck Group (2 mentions)
Millicell cm culture plate insert, by Merck Group (2 mentions)
Millicell cm insert, by Merck Group (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Collagen coated plate, by Corning (1 mentions)
Anti amcase, by Santa Cruz Biotechnology (1 mentions)
Glutamine, by Merck Group (1 mentions)
Hank s balanced salt solution hbss, by Merck Group (1 mentions)
Earle s salt, by Merck Group (1 mentions)
Millicell cm culture insert, by Merck Group (1 mentions)
Heat inactivated horse serum, by Merck Group (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
20 protocols using gey s balanced salt solution
1
Culturing E6.5 Retina Explants In Vitro
2
Tissue Collection for Neurotrauma Studies
3
Isolation and Culture of Primary Hepatocytes and HSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hepatocytes and HSCs were isolated and cultured as previously described.21 (link) Briefly, mice were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally) and the portal vein was cannulated. The livers were perfused with ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid solution (5.4 mmol/L KCl, 0.44 mmol/L KH2PO4, 140 mmol/L NaCl, 0.34 mmol/L Na2HPO4, 0.5 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 25 mmol/L Tricine, pH 7.2) followed by 0.075% type I collagenase (Sigma) in Gey's Balanced Salt Solution (Sigma). After an additional digestion step with 0.009% collagenase at 37°C agitation for 30 minutes, hepatocytes were separated with 25g centrifugation for 5 minutes at room temperature. The precipitated hepatocytes were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS) on collagen-coated plates from Corning (Corning, NY). The supernatant was centrifuged further at 400g for 10 minutes at 4°C. The cell pellet was suspended in 11.5% OptiPrep and loaded carefully with Gey's Balanced Salt Solution. After centrifugation at 1400g for 20 minutes at 4°C, the interface fraction was collected and further washed with Gey's Balanced Salt Solution twice. The finally collected HSCs were cultured further in RPMI-1640 medium containing 10% FBS and 10% horse serum.
4
Protein Extraction from Periodontal Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immediately after sample collection, periodontal tissues were washed with phosphate-buffered saline (PBS) five times and cut into pieces with a sterilized scissor. Tissue pieces were washed with Gey’s balanced salt solution (Sigma) for 30 min at room temperature to remove erythrocyte. Then, tissue pieces were lysed in an ice-cold lysis buffer (1 mM phenylmethylsulfonyl fluoride (PMSF), 0.25% sodium deoxycholate, 1 mM Na3VO4, 1% NP-40, 1 μg/mL pepstatin, 1 μg/mL leupeptin, 1 mM EDTA, 5 μg/mL aprotinin, 20 mM NaF, 150 mM NaCl, 50 mM Tris-HCl), and kept on ice for 1 h. Tissue pieces were centrifuged at 13,200 rpm at 4 °C for 10 min. Protein separation was performed by 10% SDS-PAGE. Membrane was transferred in Polyvinylidene difluoride membrane and blocked (5% non-fat skim milk) in Tris-buffered saline with 0.25% Tween 20 (TBST) at 16 °C for 1 h. The diluted anti-AMCase (Santa Cruz, CA, USA) was incubated for 18 h at 4 °C and was diluted as 1:2000–1:3000 in 5% skim milk in TBST. The membrane was incubated by secondary antibodies in 5% skim milk (1:2500–1:3000) for 2 h. Blots were then developed by Enhanced Chemiluminescence blot detecting agent (Amersham Biosciences, Piscataway, NJ, USA).
5
Organotypic Hippocampal Slice Culture Preparation
6
Retinal Explant Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The enucleated eyes were transferred to a Petri dish containing Gey’s balanced salt solution (Sigma, St. Louis, USA) supplemented with 5 mg/ml glucose (Sigma) and 50 IU-μg/ml penicillin-streptomycin (Invitrogen, Paisley, UK). Explants (about 3 mm in diameter) containing the central part of each retina were placed on membrane culture inserts (Millicell CM, Millipore, Bedford, MA, USA; pore size 0.4 μm) in 6-well plates (vitreal side downward) and cultured for two days in the culture medium under previously described conditions [31 (link)]).
7
Organotypic Hippocampal Slice Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organotypic hippocampal slice culture (OHSC) is performed as described in previous literature41 (link)52 (link) and the protocol is approved by IACUC at Columbia University. All experiments are performed in accordance with the relevant guidelines and regulations. In brief, Sprague Dawley rat pups are decapitated at postnatal day 8–10 (P8–P10), and the hippocampus is quickly isolated and placed in ice-cold Gey’s balanced salt solution (Sigma). A McIlwain tissue chopper is used to cut the hippocampus into 400 μm thick sections which are immediately plated on Millicell cell culture inserts (Millipore) or silicone MEMbranes in Neurobasal (Invitrogen) medium supplemented with B27 (1X, Invitrogen), GlutaMAX (1 mM, Invitrogen), and D-glucose (4.5 mg/ml, Sigma) at 37 °C and 5% CO2. After 2 days in vitro (DIV), the medium is changed to medium containing serum comprised of 50% MEM (Sigma), 25% heat-inactivated horse serum (Sigma), 25% Hank’s balanced salt solution (Sigma), GlutaMAX (1 mM, Invitrogen), and D-glucose (4.5 mg/ml, Sigma). Medium is changed every 2–3 days.
8
Cytotoxicity Evaluation of MTP Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The potential toxicity of MTP peptides was assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. RAW264.7 or BV-2 cells were seeded at 5000 cells per well in 100 μL of medium in a 96-well plate. The conditions tested were DMSO at a concentration of 0.5% and each peptide, at a concentration of 10−7 M. The plate was then incubated at 37 °C for the desired exposure time (4 h or 24 h). A 5 mg/mL MTT solution (Sigma) was diluted 1/50 with GBSS (Gey’s Balanced Salt Solution, Sigma). After removing the medium from each well, 100 μL of this MTT-GBSS solution was added per well. The plate was then incubated for 4 h at 37 °C. Then, 100 μL of isopropanol (VWR chemicals) was added to dissolve the purple crystals formed by the MTT reagent. The mixture in each well was aspirated and refilled to allow complete dissolution of the crystals. Finally, the optical density of each well of the plate is read by spectrophotometry (MultiskanGo, ThermoFisher Scientific) at 570 nm.
9
Culturing Retinal Explants In Vitro
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Explants from E8 retinas were cultured in vitro on 30-mm Millicell CM culture plate inserts (Millipore, Billerica, MA; pore size 0.4 µm) according to the method described by Stoppini et al. (1991) [53] with some modifications [54] (link). Retinas were dissected out into cold Gey's balanced salt solution (Sigma, St. Louis, MO) supplemented with 5 mg/mL glucose (Sigma) and 50 IU-µg/mL penicillin-streptomycin (Invitrogen, Paisley, United Kingdom). After removing the pigment epithelium, square explants (3 mm×3 mm) were isolated from the central area of each retina and then placed on Millicell inserts (Millicell CM, Millipore, Bedford, MA, USA; pore size 0.4 µm), vitreal surface down. Millicell inserts were put in six-well plates containing 1 mL/well culture medium composed of 50% basal medium with Earle's salts, 25% Hank's balanced salt solution, 25% horse serum, 1 mM L-glutamine, 10 IU-µg/mL penicillin-streptomycin (all purchased from Invitrogen), and 5 mg/mL glucose. E8 retina explants were then incubated at 37°C in a humidified atmosphere with 5% CO2 for 1 hour in vitro (hiv) to 24 hiv (E8+1hiv to E8+24hiv) according to the aim of each experiment.
10
Microcontact Printing of Protein Patterns
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microcontact printing (μCP) was used to transfer the protein structures onto a cell-repellent glass surface. This repellent behavior was achieved by silanizing with FOTCS at 45 mbar for 1.5 h in an argon atmosphere. Prior to stamping, the glass coverslips were sterilized with UV for 15 min, and the POP stamps were cleaned and sterilized in 70% ethanol in an ultrasonic bath for 15 min. To incubate the stamps in an inking solution [10 μg/mL FITC labeled poly-l -lysine (PLL) and 5 μg/mL laminin diluted in Gey’s Balanced Salt Solution (Sigma, Germany)], they were dried in a nitrogen stream and immersed in the solution structures face down. After 20 min of incubation, the stamps were entirely dried in a nitrogen stream and gently pressed onto the silanized glass coverslip for 2 min. The substrates were stored at 4°C in the dark prior to cell culture.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!