OPCs were seeded in 384-well plates (PerkinElmer, 6057500) pre-coated with poly-D-lysine and laminin (Sigma, L2020) at a density of 12,500 cell per well and allowed to attach for 1 hour at 37°C. Cell death inhibitors quinoline-Val-Asp-Difluorophenoxymethylketone (QVD-OPH) (Selleck, S7311), ferrostatin-1 (Selleck, S7243), and necrostatin-1 (Selleck, S8037), were added using a Janus automated workstation and 50 nL solid pin tool attachment in 8-point dose response (80 nM to 10 µM), and incubated for 1 hour at 37°C. Methyltrioctylammonium chloride or tributyltetradecylphosphonium chloride was added to all wells at IC90 concentrations (approximately 100 nM), and oligodendrocytes were allowed to develop for 72 hours. Negative control wells contained only methyltrioctylammonium chloride or tributyltetradecylphosphonium chloride. Positive control wells contained vehicle (DMSO). Cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, HP1–100Kit) and stained with and DAPI (1 μg/mL, Sigma, D8417). Imaging was performed with the Operetta High Content Imaging and Analysis system (PerkinElmer) and the PerkinElmer Harmony and Columbus software was used to quantify DAPI-positive nuclei.
Poly d lysine and laminin
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
Poly-d-lysine and laminin are common lab equipment used in cell culture applications. Poly-d-lysine is a positively charged polymer that enhances cell adhesion, while laminin is a protein found in the extracellular matrix that promotes cell attachment and differentiation. These products are often used to coat cell culture surfaces to improve cell growth and behavior.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (4 mentions)
6 well plate, by Corning (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Nerve growth factor (ngf), by Thermo Fisher Scientific (2 mentions)
Necrostatin 1, by Selleck Chemicals (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
D8417, by Merck Group (1 mentions)
Ferrostatin 1, by Selleck Chemicals (1 mentions)
Operetta high content imaging and analysis system, by PerkinElmer (1 mentions)
Harmony and columbus software, by PerkinElmer (1 mentions)
Incubator shaker, by Eppendorf (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Neurobasal a, by Thermo Fisher Scientific (1 mentions)
Collagenase from clostridium histolyticum, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Worthington (1 mentions)
Papain, by Worthington (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
18 protocols using poly d lysine and laminin
1
High-Throughput Oligodendrocyte Viability Assay
2
Primary Cortical Neuron Culture and Treatments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The culture of primary cortical neurons was carried out as previously described [22 (link)]. Briefly, the cerebral cortexes of 1-day-old neonatal rats were harvested, homogenated and culture. The complete culture medium composed of DMEM/HIGH GLUCOSE, 10% fetal calf serum and 1% penicillin–streptomycin solution as used for cell culture. Neurons were plated in 6-well plates (Corning, United States) coated with poly-d -lysine and laminin (Sigma-Aldrich, St. Louis, MO, United States) at a density of 2–5 × 105/ml, and incubated at 37 °C, 5% CO2. Moreover, the complete medium was replaced by neuron specific medium (neuron basal + 2% B27, no serum) (Invitrogen, Carlsbad, CA). The culture medium was then changed the next day. Neurons were divided into 4 groups (n = 3 well/group): normal group (neurons without any treatment); OGD group (Neurons were subjected to OGD); Control group (OGD neurons received 1/3000 DMSO); OGD + Scu 3 μM group (OGD neurons received 3 μM Scu administration).
3
Preparation of Primary Nodose Ganglion Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary nodose ganglion neuronal cultures were prepared from adult female rats as described previously with some modifications [30 (link)]. Briefly, rats were deeply anesthetized with overdose of isoflurane. The nodose ganglia of the vagus nerve were carefully removed and washed in Hank’s balanced saline solution (HBSS) (Invitrogen) (in mM: 137 NaCl, 5.4 KCl, 0.4 KH2PO4, 1 CaCl2, 0.5 MgCl2, 0.4 MgSO4, 4.2 NaHCO3, 0.3 Na2HPO4, and 5.6 glucose). The ganglia were then incubated on an incubator shaker (New Brunswick Scientific, NJ, USA) for 30 min at a speed of 135 rpm at 37 °C in HBSS containing papain (15 U/ml, Worthington Biochemical, NJ, USA) and collagenase from clostridium histolyticum (5 × 10−4 g/ml, Sigma), rinsed three times with HBSS, and placed in culture medium containing Neurobasal A (Invitrogen), 2% B-27 (Life Technologies, NY, USA), 2% heat-inactivated horse serum, 2% fetal calf serum, 0.2 mM L-glutamax, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The fragments were mechanically dissociated by gently triturating with a pipette. The dispersed cells were seeded onto 12-mm poly-D-lysine and laminin (Sigma)-coated coverslips at a density of 3000 cells per well. Neurons were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
4
Purification and Culture of Retinal Ganglion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary RGCs were isolated and purified by immunopanning.25 Briefly, the retinas of 3‐day‐old SD rats were triturated and digested with 5 mg/mL papain. The dissociated cell suspension was incubated on a panning plate coated with goat anti‐rabbit IgG to remove macrophages. Next, the Thy1.1‐positive RGCs were purified using a second panning plate coated with goat anti‐mouse IgM and mouse anti‐Thy1.1 antibodies (Invitrogen). The detached RGCs were seeded at a density of ~ 1.5 × 104/mm2 and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), BDNF (50 ng/mL; Peprotech), CNTF (10 ng/mL; Peprotech), and forskolin (5 ng/mL; Sigma‐Aldrich) in culture slides precoated with poly d ‐lysine and laminin (Sigma‐Aldrich). For survival assays, SP1‐shRNA was transfected into cells using Lipofectamine RNAiMAX as recommended by the manufacturer. At 48 hours after transfection, the cells were treated with or without recombinant Nogo‐66 (50 μg/mL; R&D Systems) or LINGO‐1 (100 μg/mL; LifeSpan) in the presence of absence of SP1‐shRNA. After incubation for 12 hours, complete medium was replaced with DMEM without serum to induce apoptosis. Cell cultures with serum were performed in parallel as controls.
5
Primary Neuron Isolation from Mouse Cortex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary neuron cells were isolated from the cerebral cortex of 16-day C57BL/6 mouse embryos as described previously [70 (link)]. Briefly, the cerebral cortex was separated from the brains and then dissociated with 0.25% trypsin (wt/vol) at 37°C for 30min. After centrifugation, the cells were resuspended in DMEM containing 5% FBS (Gibco) and subsequently passed through a 70 μm filter. After cell counting, the cells were seeded into 12-well plates (SORFA Life Science Co., Ltd, Beijing, China) that had been pretreated with poly-D-lysine and laminin (10 μg/ml) (Sigma, P7280). The cell supernatants were discarded at 5 h after seeding and replaced with Neurobasal plus Medium (Gibco, A3582901) containing 2% B-27 Plus (Gibco, A3582801). Then replace the medium every two days until 5–6 days post seeding for experiments.
6
Dorsal Root Ganglia Isolation and Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DRGs (C1-L6) from WT and FcRγ chain−/− mice were extracted and placed in ice-cold Dulbecco’s PBS until enzymatically dissociated with papain (1.7 mg/ml; 30 min at 37°C) followed by treatment with collagenase I (2 mg/ml) and dispase II (8 mg/ml; Sigma-Aldrich) enzyme mixture (30 min at 37°C). The cells were then gently triturated in Leibovitz’s medium (L15) supplemented with 10% heat-inactivated FBS, 1% penicillin and streptomycin (Invitrogen), and 10 µM mitotic inhibitor (5-fluoro-2-deoxyuridine; Sigma-Aldrich). For CGRP release experiments, nerve growth factor (30 ng/ml; Sigma-Aldrich) was added into the medium. The cell suspension was plated on uncoated well plates for 1.5–2 h before being transferred to well plates precoated with poly-d -lysine and laminin (Sigma-Aldrich). The cells were maintained at 37°C in 5% CO2 atmosphere, and the medium was replaced after 24 h, followed by changes every third day.
7
Isolation and Culture of Primary Cortical Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary cortical neurons were separated from neonatal SD rats within 1 day of birth. The obtained small cortical tissue was placed in a culture ware containing 2 mL DMEM (high-glucose) liquid. Cortices were harvested and cut into approximately 1-mm3 small pieces, then digested with 0.05% trypsin (Gibco, Rockville, MD, United States) at 37°C for 10 min. Then, the high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) was added to the culture ware to stop digestion. The tissue suspension was centrifuged at 1000 rpm for 10 min, resuspended, and plated in 96-well or 6-well plates (Corning, Corning, NY, United States) coated with poly-d-lysine and laminin (Sigma-Aldrich, St. Louis, MO, United States) at a density of 5 × 105 cells/ml with a neurobasal medium. After this, half of the neurobasal medium was refreshed every 3 days.
8
Isolation and Culture of Primary Cortical Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary cortical neurons were obtained from SD rats at 1‐day postnatal. Briefly, the cortices of the rats were harvested and sectioned into approximately 1 mm3 pieces. Subsequently, the tissue sections were digested with 0.25% trypsin (Gibco) at 37°C for 10 min and eluted with 10% fetal bovine serum (FBS, BSA, Gibco). The samples were centrifuged at 1500 rpm for 5 min, and the pellets were resuspended using a Hyclone medium containing Dulbecco's modified Eagle medium (DMEM)/HIGH GLUCOSE, 10% FBS, and 1% penicillin‐streptomycin solution). Next, the neurons were seeded at a density of 5 × 105 cells/ml into 6‐well plates (Corning, USA) coated with poly‐d ‐lysine and laminin (Sigma‐Aldrich), then incubated at 37°C with 5% CO2. The completed medium was replaced with 2% B27 (Invitrogen, CA) at 4 h after the incubation. The medium was changed the next day and then half change was made every 3 days. PC12 cells were obtained from American Type Culture Collection and cultured in DMEM (Cat. No. 10569‐010) supplemented with 10% FBS (Cat. No. 16000‐077). Cortical neurons were subjected to oxygen‐glucose deprivation (OGD) insult as previously described.22
9
Oligodendrocyte Differentiation Assay
10
Isolation and Co-culture of Astrocytes and Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary rodent astrocytes were isolated from the cerebral cortex of E18.5 CD 1 mice as described elsewhere [25 (link)]. Briefly, cortices were dissected, enzymatically digested and mechanically dissociated. Astrocytes were plated on poly-d -lysine and laminin (Sigma)-coated plates in medium containing DMEM supplemented with 10% FBS (Thermo Fisher Scientific) and passaged twice prior to co-culturing with neurons on a 24-well plate. For co-cultures, human NPCs (30,000 cells/coverslip) were plated on to a layer of astrocytes and differentiated into neurons in a humidified incubator (5% CO2) at 37 °C for 8 weeks.
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!