Human embryonic kidney 293 (HEK293) cells stably expressing hNav1.7 were cultured in high-glucose Dulbecco's Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and were selected with a 300 μg/mL solution of the antibiotic hygromycin B (Invitrogen, Carlsbad, CA, USA) under standard tissue culture conditions (5% CO2, 37 °C). Human Nav1.5 channels were stably expressed in Chinese hamster lung (CHL) cells that were cultured in high-glucose DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and were selected with a 500 µg/mL solution of the antibiotic G418 (Invitrogen, Carlsbad, CA, USA). Dorsal root ganglion (DRG) neurons were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Before electrophysiological recording, all cells were plated onto poly-L-lysine-coated glass coverslips (Sigma-Aldrich, St Louis, MO, USA) and used within 24 h.
Poly l lysine coated glass coverslips
Manufactured by Merck Group
Sourced in United States
Poly-L-lysine-coated glass coverslips are a type of laboratory equipment used for various cell culture and microscopy applications. They provide a positively charged surface that enhances the adhesion and attachment of cells to the substrate.
Automatically generated - may contain errors
Lab products found in correlation
35 mm culture dishes, by Scientific Laboratory Supplies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Roller tubes, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Leica dmi6000b, by Leica Microsystems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Leica Microsystems (1 mentions)
Ab152, by Merck Group (1 mentions)
Alexa fluor 546 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Sc 23927, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Hek293a, by Thermo Fisher Scientific (1 mentions)
11 protocols using poly l lysine coated glass coverslips
1
Cell Culture Protocol for Voltage-Gated Sodium Channels
2
Neocortical Slice Culture from Mice
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All animal protocols were approved by the Massachusetts General Hospital Center for Comparative Medicine. Neocortical slices were prepared at postnatal day 6–7 from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) and from Clomeleon mice. Clomeleon mice express a genetically encoded Cl− ratiometric fluorophore.49 Slice cultures were prepared as described for hippocampal organotypic cultures.37 , 50 Briefly, coronal 400‐μm‐thick neocortical slices (frontal region, prehippocampus) were cut using a McIlwain tissue chopper (Mickle Laboratory Eng. Co., Surrey, U. K.; Fig. 1 A). Slices were mounted in clots of chicken plasma (Cocalico Biologicals, Reams‐town, PA) and thrombin on poly‐l ‐lysine‐coated glass coverslips (Sigma‐Aldrich, St. Louis, MO). Slices were incubated in roller tubes (Nunc, Roskilde, Denmark) at 36°C with 750 μL of Neurobasal A/B27 medium supplemented with 0.5 mM GlutaMAX, 30 μg/mL gentamicin (Life Technologies, Grand Island, NY) and 20 mM NaCl to reach a final media osmolarity of 280 ± 5 mOsm (mean ± SD, n = 7). The culture media were changed biweekly.
3
Cellular Uptake and DOXO Release
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Particle uptake and DOXO release inside cells
were also followed by fluorescence microscopy. HeLa cells were seeded
on poly-l -lysine coated glass coverslips (12 × 12 mm2, Sigma-Aldrich) placed inside 6-well plates (2 × 105 cells per well) with 2 mL of DMEM and grown for 24 h at standard
culture conditions. Then, PSS/PLL/DOXO/HA-coated and (PSS/PLL/DOXO)2/HA-coated GNRs (2.5 × 1010 NP/mL, 250 μL)
were added to cells. After 6 h of incubation, the cells were washed
three times with PBS and fresh medium was added. Next, some cells
were irradiated with a CW 808 nm fiber-coupled diode laser source
(50 W, Oclaro Inc., San Jose, CA) for 5 min at 0.5 W/cm2. After the desired incubation time (4, 6, 8, 12, and 24 h), the
cells were washed three times with PBS, fixed with 4% (w/v) paraformaldehyde
for 10 min, washed again with PBS, treated with Triton X-100 for 10
min, and, finally, washed again with PBS. Then, the coverslips were
mounted on glass slides, stained with DAPI (Invitrogen, USA) and cured
for 24 h at −20 °C. The samples were visualized at 63×
using a wide field fluorescence inverted microscope (Leica DMI6000B,
Leica Microsystems, Germany) using the blue channel for DAPI (λex = 350 nm), the red channel for DOXO (λex = 520 nm), and transmitted light in differential interference contrast
mode.
were also followed by fluorescence microscopy. HeLa cells were seeded
on poly-
culture conditions. Then, PSS/PLL/DOXO/HA-coated and (PSS/PLL/DOXO)2/HA-coated GNRs (2.5 × 1010 NP/mL, 250 μL)
were added to cells. After 6 h of incubation, the cells were washed
three times with PBS and fresh medium was added. Next, some cells
were irradiated with a CW 808 nm fiber-coupled diode laser source
(50 W, Oclaro Inc., San Jose, CA) for 5 min at 0.5 W/cm2. After the desired incubation time (4, 6, 8, 12, and 24 h), the
cells were washed three times with PBS, fixed with 4% (w/v) paraformaldehyde
for 10 min, washed again with PBS, treated with Triton X-100 for 10
min, and, finally, washed again with PBS. Then, the coverslips were
mounted on glass slides, stained with DAPI (Invitrogen, USA) and cured
for 24 h at −20 °C. The samples were visualized at 63×
using a wide field fluorescence inverted microscope (Leica DMI6000B,
Leica Microsystems, Germany) using the blue channel for DAPI (λex = 350 nm), the red channel for DOXO (λex = 520 nm), and transmitted light in differential interference contrast
mode.
4
Quantifying Nestin and Tyrosine Hydroxylase Immunofluorescence
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Cells seeded on poly-L-lysine coated glass coverslips (Sigma Aldrich) were fixed after treatment with 4% paraformaldehyde and stained with primary mouse anti-nestin antibody (sc-23927, Santa Cruz, 1:50) and with rabbit anti-TH (Tyrosine hydroxylase) antibody (AB152, Sigma-Aldrich, 1:250). Nuclei were stained with DAPI (Merck, Kenilworth, NJ, USA). Donkey anti-mouse Alexa Fluor 546 or donkey anti-rabbit Alexa Fluor 488 secondary antibodies were used (Thermo Fisher, Waltham, MA, USA). Fluorescence intensity was quantified using ImageJ software. Values are indicated as normalized corrected total cell fluorescence (CTCF) expressed as arbitrary units (A.U.).
5
Glycine Receptor Expression in Cell Lines
6
Immunofluorescence Characterization of Mesenchymal Cells
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Hamster-PSCs of P2 passage, cancer-associated fibroblasts of P3 passage, and rat-PSCs of P3 passage were seeded on poly L-lysine-coated glass coverslips (Sigma) in six-well plates. After achieving the minimum required confluence, cells were washed with PBS twice for five minutes per wash, followed by fixation with an ice-cold mixture of methanol and acetone (1:1 ratio) for 10 minutes at −20°C; nonspecific binding was blocked with 2% BSA (MP Biomedical) in PBS for 30 minutes. Cells were then incubated with an anti-α-SMA primary antibody (1:100) overnight in a humidified chamber at 4°C, followed by anti-mouse IgG-ALEXA FLOUR 594 (1:500) for 45 minutes at room temperature. Finally, cells were washed with PBS and mounted with mounting medium containing DAPI (Life Technologies) to stain the nuclei. Stained slides were examined under a Leica DMIL LED microscope, and images were captured at 10× and 20× magnifications.
7
HEK 293 Cell Transfection Protocol
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HEK 293 cells (from American Type Culture Collection) were grown at 37 °C in a humidified 95% air, 5% CO2 incubator in DMEM (Gibco, 41966029) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml of penicillin G, 100 μg/ml of streptomycin sulfate (all from Invitrogen). Cells were passaged after reaching 70–80% confluence every 2–3 days, up to 25 times.
For expression, cells were plated on poly-l -lysine–coated glass coverslips (Sigma-Aldrich and VWR, respectively) in 35-mm culture dishes (Scientific Laboratory Supplies) containing 2 ml of DMEM, and then transfected via the calcium phosphate-precipitation method (58 (link)) with pcDNA3 plasmids coding for the above mentioned GlyRs.
A plasmid coding for the enhanced green fluorescent protein was added to allow detection of transfected cells. The final DNA mixture contained 2% GlyR cDNA, 20% enhanced green fluorescent protein cDNA, and 78% empty pcDNA3 plasmid. The total amount of the final DNA mixture was 3 μg/plate. The transfection medium was washed off and replaced by fresh medium 4–8 h after transfection. Electrophysiological experiments were performed 1–2 days after transfection.
For expression, cells were plated on poly-
A plasmid coding for the enhanced green fluorescent protein was added to allow detection of transfected cells. The final DNA mixture contained 2% GlyR cDNA, 20% enhanced green fluorescent protein cDNA, and 78% empty pcDNA3 plasmid. The total amount of the final DNA mixture was 3 μg/plate. The transfection medium was washed off and replaced by fresh medium 4–8 h after transfection. Electrophysiological experiments were performed 1–2 days after transfection.
8
Analyzing RipA-Mediated Mitochondrial Dynamics
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HEK293T cells were grown in 24-well-tissue culture plates on sterile poly-L lysine coated glass coverslips (Sigma, USA). Cells were transfected with pcDNA3.1+, pcDNA3.1+-RipA, EGFP-N1, or EGFP-N1-RipA using Lipofectamine 3,000 Transfection Reagent (ThermoFisher Scientific, USA). After 24, 48, and 72 h of transfection, cells were treated with 300 nm of MitoTracker Deep Red FM (ThermoFischer Scientific, USA) for 45 min in incomplete DMEM. In another experiment, RAW264.7 cells were treated with RipA (0.5, 1, and 2 μg/ml) for 24 h. After fixation and permeabilization, cells were blocked and coverslips were incubated with anti-RipA (1:500), and various other primary antibodies (anti-LC3B, anti-NFkB, anti-CoxIV, and anti-TLR4) as desired. Alexa Fluor 488 and Alexa Fluor 594-conjugated secondary antibodies were added at 1:500 dilution for 2 h at room temperature. Finally, coverslips were sealed with ProLong Glass Antifade mountant (Thermo Fisher, USA). Samples were visualized at 60 and 63X through Nikon's Confocal microscope and Carl Zeiss Fluorescence microscope equipped with oil immersion objectives. Images were captured and processed using NIS Elements 5.21.00 and an Axio Cam Hrm digital camera and Axio-Vision-4.8 software. Punctate foci representing LC3 were counted manually from 10 different fields and data presented are a representative count from 50 cells.
9
Fluoxetine and Stem Cell Proliferation
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NPCs were plated in six-well plates at a density of 1×106 cells per well and cultured in the presence of different concentrations of fluoxetine, CHIR99021, XAV939 (XAV), or WAY-100635 as indicated in the figures. After 2 d, 5’-bromo-2-deoxy-uridine (BrdU; 10 μM, Sigma) was added, and after a further incubation for 24h, the cells were dissociated and plated onto poly-L-lysine-coated glass coverslips (Sigma). After attachment, NPCs were fixed for 20min in 4% paraformaldehyde and treated with 2M HCl for 30min at room temperature. Cells were washed with PBS and incubated overnight with rat anti-BrdU (1:200, Abcam) at 4°C. After washing in PBS, the cells were incubated with rhodamine-conjugated rabbit anti-rat lgG (1:100, Jackson ImmunoResearch) for 1h at room temperature. Labeled cells were further incubated with DAPI (Sigma) at 0.1 μg/mL for 30min at room temperature prior to mounting with Gel Mount. The numbers of total cells and BrdU-positive cells were counted using fluorescence microscopy in four non-overlapping fields per coverslip. Cells incubated without the primary antibody served as a negative control.
10
Isolation and Culturing of Primary Neuronal Cells
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