24 h prior to transfection, 293T cells were seeded at 65–75% confluency into 24-well plates coated for 30 min with a 1 mg/mL solution of poly-D-lysine (MP Biomedicals Inc.) and washed with PBS (Gibco) once prior to media addition. The next day, 500 ng of 3CLpro expression plasmid, unless otherwise stated, was incubated with Opti-MEM and Lipofectamine 2000 for 30 min at room temperature prior to dribbling on cells, as per manufacturer’s protocol. For plating into drug conditions, 20 h after transfection, cells were washed once with PBS and 200 μL Trypsin-EDTA 0.25% (Gibco) was added to cells to release them from the plate. Trypsinized cell slurry was pipetted up and down repeatedly to ensure a single cell suspension. 96-well plates were coated with poly-D-lysine, either coated manually with 1 μg/mL poly-D-lysine in PBS solution for 30 min or purchased pre-coated with poly-D-lysine (Corning). Wells were filled with 100 μL of media ± drug and 1 μg/mL puromycin and were seeded with 9 μL of trypsinized cell slurry. For higher throughput experiments, multiple individually transfected wells of a 24-well plate were combined after trypsinization and prior to seeding in drug. After seeding into wells containing drug and puromycin, cells were incubated for 48 h unless otherwise specified.
Poly d lysine
Manufactured by Corning
Sourced in United States
Poly-D-lysine is a synthetic polymer used as a cell culture surface coating to promote cell adhesion and growth. It provides a positively charged substrate that enhances the attachment of cells to the culture surface.
Automatically generated - may contain errors
Lab products found in correlation
Laminin, by Corning (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Neurobasal medium, by Thermo Fisher Scientific (6 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Hoechst 333242, by Thermo Fisher Scientific (4 mentions)
Vectashield mounting medium, by Vector Laboratories (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Papain, by Worthington (4 mentions)
Matrigel, by Corning (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Glutamine, by PanEco (3 mentions)
Ventilated culture vials, by Corning (3 mentions)
D glucose, by Biosera (3 mentions)
Fetal bovine serum (fbs), by Biosera (3 mentions)
Dulbecco modified eagle medium (dmem), by PanEco (3 mentions)
Neurobasal a, by Thermo Fisher Scientific (3 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
62 protocols using poly d lysine
1
Transient 3CLpro Expression in 293T Cells
2
Isolation and Culture of Astroglial and Neuroglial Cells
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Astroglial cell cultures were isolated from the brains of 1–2-day-old mouse according to the modified McCarthy and de Vellis protocol [87 (link)]. The brains were extracted, the cerebral cortex was separated, the meninges were removed, and the tissue was minced and incubated in 0.05% trypsin-EDTA solution at 37 °C for 30 min. After enzymatic digestion, the tissues were washed twice in PBS and then dissociated by glass Pasteur pipette in a culture medium consisting of DMEM (PanEco, Moscow, Russia), 1 g/L D-glucose, and 10% FBS (Biosera, Kansas City, MO, USA), with the addition of 2 mM glutamine (PanEco, Russia). The suspension of cells was transferred on ventilated culture vials (Costar, Washington, DC, USA) precoated with poly-D-lysine (10 μg/mL). The cells were cultivated at 37 °C and 5% CO2. After 5–6 days, the cultures were shaken on an orbital shaker at 200 rpm for 16 h to detach and remove microglia. After 10 to 20 days, in vitro astrocytes were used for experiments.
Neuroglial cortical cultures were prepared in a similar manner as astroglial cultures, but cells were cultured in Neurobasal-A medium containing glutamine, B-27 (2%, Thermo Fisher Scientific, Waltham, MA, USA, RRID: CVCL_A315) and gentamicin (20 μg/mL, Sigma-Aldrich, St. Louis, MI, USA, Cat #G1397) [25 (link)].
Neuroglial cortical cultures were prepared in a similar manner as astroglial cultures, but cells were cultured in Neurobasal-A medium containing glutamine, B-27 (2%, Thermo Fisher Scientific, Waltham, MA, USA, RRID: CVCL_A315) and gentamicin (20 μg/mL, Sigma-Aldrich, St. Louis, MI, USA, Cat #G1397) [25 (link)].
3
Isolation and Culture of Astroglial Cells
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Astroglial cell cultures were isolated from the brains of 1–2-day-old rats according to the modified McCarthy and de Vellis protocol [87 (link)]. The brains were extracted, the cerebral cortex was separated, the meninges were removed, and tissue was ground and incubated in 0.05% trypsin-EDTA solution at 37 °C for 30 min. After enzymatic digestion, the tissues were washed twice in PBS and then dissociated by glass Pasteur pipette in a culture medium consisting of DMEM (PanEco, Russia), 1 g/L D-glucose, and 10% FBS (Biosera, Kansas City, MO, USA), with the addition of 2 mM glutamine (PanEco, Moscow, Russia). The suspension of cells was transferred on ventilated culture vials (Costar, Washington, DC, USA) precoated with poly-D-lysine (10 µg/mL). The cells were cultivated at 37 °C and 5% CO2. After 5–6 days, the cultures were subjected to vibration on an orbital shaker at 200 rpm for 16 h to detach and remove microglia. After 10 to 20 days, in vitro astrocytes were used for experiments.
4
Astroglial Cell Culture from Neonatal Rat Brains
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Astroglial cell cultures were isolated from the brains of 1–2-day-old rats according to the modified McCarthy and de Vellis protocol [67 (link)]. The brains were extracted, the cerebral cortex was separated, the meninges were removed, and tissue was ground and incubated in 0.05% trypsin-EDTA solution at 37 °C for 30 min. After enzymatic digestion, the tissues were washed twice in PBS and then dissociated by glass Pasteur pipette in a culture medium consisting of DMEM (PanEco, Russia), 1 g/L D-glucose, and 10% FBS (Biosera, Kansas City, MO, USA), with the addition of 2 mM glutamine (PanEco, Russia). The suspension of cells was transferred on ventilated culture vials (Costar, Washington, DC, USA) precoated with poly-D-lysine (10 mcg/mL). The cells were cultivated at 37 °C and 5% CO2. After 5–6 days, the cultures were subjected to vibration on an orbital shaker at 200 rpm for 16 h to detach and remove microglia. After 10 to 20 days, in vitro astrocytes were used for experiments.
5
Nucleoside Uptake and Nucleolar Localization
6
Nucleoside Uptake and Nucleolar Localization
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Coverslips in 6-well tissue culture plates were treated with 1× poly-D lysine (Corning) solution for 3 h at 37 °C. HEK293T cells were seeded at densities of 2.5×105 and grown to ~50% confluency on glass cover slips. Cells with or without ectopic expression of UCK2 were treated with nucleosides to final concentrations of 500 μM and incubated for 5 hours. After labeling, cells were washed twice with DPBS and fixed for 10 min at room temperature with 3.7% paraformaldehyde. Cells were quenched with 50 mM glycine, DPBS for 5 min, and then washed twice more. Cells were permeabilized in 0.1% Triton-X DPBS for 30 min, and then washed ×2 with DPBS. HEK293T cells were blocked with 10% goat serum/DPSB for 2 h. Cells were than incubated with 1:500 anti-NPM1 (anti-B23; Sigma Aldrich) in 1% BSA/DPBS overnight at 4 °C, followed by washing ×3 with DPBS. Cells were then incubated with 1:1000 anti-rabbit Alexa 647 (Fisher Scientific) in 1% BSA/DPBS for 1 h in the dark at room temperature. Cells were stained with a solution of 1:3000 Hoechst 333242 for 10 min (Thermo Fisher Scientific). Coverslips were briefly washed and then mounted using VectaShield mounting medium (Vector Labs). Slides were imaged via fluorescence confocal microscopy using a 63× oil immersion objective on a Leica 700 Carl Zeiss microscope.
7
Clickable Nucleoside Incorporation Assay
8
Clickable Nucleoside Incorporation Assay
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Coverslips in 6-well tissue culture plates were treated with 1× poly-D lysine (Corning) solution for 3 h at 37 °C. HEK293T cells were seeded at densities of 2.5×105 and grown to ~50% confluency on glass cover slips. Cells with or without ectopic expression of UCK2 were treated with nucleosides to final concentrations of 1 mM and incubated for 5 hours. After labeling, cells were washed twice with DPBS and fixed for 10 min at room temperature with 3.7% paraformaldehyde. Cells were quenched with 50 mM glycine, DPBS for 5 min, and then washed twice more. Cells were permeabilized in 0.1% Triton-X DPBS for 15 min, and then washed ×2 with DPBS. Cells were then treated with solutions containing 500 μM CuSO4, 2.5 mM THPTA (Sigma), 2.5 mM sodium ascorbate, and 15 μM Alexa-Fluor 568 (Click Chemistry Tools) and incubated at room temperature for 1 h in the dark. Cells were washed ×2 with 0.1% Triton-X DPSB for 2 min, and then ×5 with DPBS for 5 min each on an orbital shaker. Cells were stained with a solution of 1:3000 Hoechst 333242 for 10 min (Thermo Fisher). Coverslips were briefly washed and then mounted using VectaShield mounting medium (Vector Labs). Slides were imaged via fluorescence confocal microscopy using a 63× oil immersion objective on a Leica 700 Carl Zeiss microscope.
9
Embryonic Mouse Cortical Neuron Culture
10
Embryonic Mouse Cortical Neuron Culture
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Primary neuronal cultures were prepared from embryonic day 15.5 Trio+/flox mouse embryos (combination of sexes). Cortices were dissected in ice–hold HBSS (Invitrogen) with 25 mM HEPES and 0.5% glucose and then digested in the same solution with 0.3 U/ml papain (Worthington, Lakewood, NJ), 0.1% dispase (Roche, Indianapolis, IN), and 0.01% DNaseI (Sigma) at 37°C (twice for 7 minutes). Tissues were triturated 20 times with a glass Pasteur pipette (Moresco et al., 2005 (link)). Neurons were resuspended and plated in Neurobasal A (Invitrogen) supplemented with 2% Gem21 (Gemini) and 10% FBS on 12 mm coverslips precoated with 20 μg/ml polyD–lysine (Corning Inc.) overnight at 37°C and 1 μg/ml laminin (Corning) for 2 hours at 37°C. After 4 hours, the media was changed to serum–free Neurobasal A/Gem21 media containing 1% penicillin/streptomycin and 2 μM L–glutamine, and cultures were maintained at 37°C and 5% CO2. Glial growth was suppressed with 1 μM ara–C (Sigma) on day in vitro (DIV) 3 and 0.5 μM ara–C on DIV6.
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