N2A Neuronal cells (ATCC CCL-131, ATCC, Manassas) were grown in 96 Well-plates and pre-activated with mIFN-γ (100 ng/ml, R&D Systems) for 24 h. Thereafter, medium was replaced after extensive washes with PBS with astrocyte-conditioned media. Cytotoxicity was measured using LDH release (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) after 24 h as suggested by manufacturer’s protocol.
Atcc ccl 131
Manufactured by American Type Culture Collection
Sourced in United States
ATCC CCL-131 is a cell line derived from the lungs of an adult male Caucasian. It is an adherent epithelial-like cell line.
Automatically generated - may contain errors
Lab products found in correlation
Cytotox 96 non radioactive cytotoxicity assay, by Promega (2 mentions)
Mifn γ, by R&D Systems (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fps zm1, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Be12 614f, by Lonza (1 mentions)
P6407, by Merck Group (1 mentions)
P6282, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by American Type Culture Collection (1 mentions)
Amaxa nucleofector system, by Lonza (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Atcc scrc 1008, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin antibiotic, by Thermo Fisher Scientific (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
12 protocols using atcc ccl 131
1
Assessing Astrocyte-Mediated Neuronal Cytotoxicity
2
Investigating Neuro-2a Cell Response to S100B
3
Culture and Maintenance of Mouse Cell Lines
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Mouse BV-2 cells (43 (link)) were cultured in RPMI-1640 medium (R7509, Sigma-Aldrich) supplemented with 2.4 mM L-glutamine (17-605E; Gibco), 10% (v/v) fetal bovine serum (10270-106; Gibco), and 1.2% (v/v) penicillin/streptomycin (15140-122; Gibco). BV-2 cells were scraped off gently from cell culture plates (168381; Thermo Scientific) and collected by centrifugation at room temperature (RT) for 3 min at 800 × g. Cell pellets were resuspended in cell culture medium and plated for maintenance or experiments on cell culture plates or poly-l -lysine (P6282; Sigma-Aldrich) coated glass coverslips. Mouse Neuro-2a (N2a) neuroblastoma cells (ATCC CCL-131; ATCC) were maintained in Dulbecco's Modified Eagle's Medium (BE12-614F; Lonza) containing 2.4 mM L-glutamine, 10% (v/v) fetal bovine serum, and 1.2% (v/v) penicillin/streptomycin. N2a cells were washed once with warm Dulbecco's phosphate-buffered saline (DPBS, 17-512F; Lonza) and incubated in 0.25% trypsin solution (in DPBS, 15090-046; Gibco) for 3–5 min at +37°C. Next, the cell suspension was diluted in the culture medium and centrifuged for 3 min at 800 × g. The supernatant was discarded, and the cells were resuspended in the culture medium and plated for experiments on poly-d -lysine (P6407, Sigma-Aldrich) coated coverslips or on cell culture plates for maintenance.
4
Neuro-2a Cell Response to Microglia-Conditioned Media
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Mouse neuroblastoma cell line Neuro-2a (N2a) (ATCC® CCL-131™) was obtained from ATCC (Manassas, VA, USA). These N2a cells were maintained on Eagle’s Minimum Essential Medium (EMEM) (ATCC® 30-2003™) supplemented with 10% FBS, 100 U/mL Penicillin, 100 µg/mL Streptomycin (Gibco, NY, USA) and incubated in a humidified 5% CO2 incubator at 37 °C.
Cells were plated on a 6 well plate with a seeding density of 0.3 × 106 cells/well and were grown till the cells reached almost 70% confluency. Then, the N2a cells were serum-starved for 18 h using EMEM supplemented with 1% FBS. Following serum starvation, the cells were treated with a combination of MCM obtained from each SIM-A9 microglia experimental group and normal N2a growth medium for 24 h. The groups designed for this experiment were unchanged from the last SIM-A9 cells experiment (CONTROL, IL-6, AG50, and IL-6+AG50). Post-treatment, cell supernatants were collected, stored at −80 °C, and proteins from each group were extracted using 1× RIPA lysis buffer containing protease and phosphatase inhibitors. The concentration of the extracted protein samples from each group was estimated using the BCA assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and the Western Blot experiment was performed using these extracted protein samples.
Cells were plated on a 6 well plate with a seeding density of 0.3 × 106 cells/well and were grown till the cells reached almost 70% confluency. Then, the N2a cells were serum-starved for 18 h using EMEM supplemented with 1% FBS. Following serum starvation, the cells were treated with a combination of MCM obtained from each SIM-A9 microglia experimental group and normal N2a growth medium for 24 h. The groups designed for this experiment were unchanged from the last SIM-A9 cells experiment (CONTROL, IL-6, AG50, and IL-6+AG50). Post-treatment, cell supernatants were collected, stored at −80 °C, and proteins from each group were extracted using 1× RIPA lysis buffer containing protease and phosphatase inhibitors. The concentration of the extracted protein samples from each group was estimated using the BCA assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and the Western Blot experiment was performed using these extracted protein samples.
5
Profiling Knockdown Techniques in Neuro-2a and MEFs
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siProfilin2-399 (5′-CAUCACGCCAGUAGAAAUATT-3′), siProfilin2-527 (5′-CAAUGGACAUCCGGACAAATT), siNCAM (5′-GUUGGAGAGUCCAAAUUCUTT-3′), and NC (5′-UUCUCCGAACGUGUCACGUTT-3′) were synthesized by GenePharma. shProfilin2 was constructed by GeneChem by inserting the same target sequence as siProfilin2-399 into the GV102 vector. To confirm siProfilin2 efficacy, Neuro-2a cells (ATCC CCL-131; American Type Culture Collection) were transfected with siRNA/shRNA using Lipofectamine 2000 reagent according to the manufacturer’s instructions (11668030; Invitrogen). MEFs (ATCC SCRC-1008; American Type Culture Collection) were transfected with siRNA using Lipofectamine 2000. The profilin2/NCAM knockdown efficacy was verified by Western blot analysis of cell lysates. Cultured NPCs were transfected with 20 pmol of RNA per cuvette using the Amaxa Nucleofector system (VPG-1004; Lonza) according to the user’s manual.
6
Assessing Astrocyte-Mediated Neuronal Cytotoxicity
7
Generating Luciferase-Expressing Neuro-2a Cells
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Neuro-2a cells (ATCC® CCL-131™) were purchased from the American Type Culture Collection (ATCC).
Cells were maintained in Eagle's Minimum Essential Medium (EMEM) (ATCC) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin antibiotic (Thermo-Fisher Scientific). Luciferaseexpressing Neuro-2a cells were generated by the transduction of cells with luciferase expression vector. The lentiviral vector pLenti CMV Puro LUC encoding firefly luciferase was transfected with Virapower Lentiviral packing mix to 293T cells (ATCC® CRL-3216™) using lipofectamine 2000 (Invitrogen). After 48 h, lentiviral supernatant was collected and added into 20-40% confluent Neuro-2a cells. Polybrene (8 μg/mL) was added during the transduction. Two days after the transduction, Neuro-2a cells were selected by puromycin (2 μg/mL) and were maintained in EMEM media containing 10% FBS and 1 μg/mL puromycin. All cells were incubated at 37 ºC in a humidified atmosphere containing 5% CO 2 in a cell culture incubator.
Cells were maintained in Eagle's Minimum Essential Medium (EMEM) (ATCC) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin antibiotic (Thermo-Fisher Scientific). Luciferaseexpressing Neuro-2a cells were generated by the transduction of cells with luciferase expression vector. The lentiviral vector pLenti CMV Puro LUC encoding firefly luciferase was transfected with Virapower Lentiviral packing mix to 293T cells (ATCC® CRL-3216™) using lipofectamine 2000 (Invitrogen). After 48 h, lentiviral supernatant was collected and added into 20-40% confluent Neuro-2a cells. Polybrene (8 μg/mL) was added during the transduction. Two days after the transduction, Neuro-2a cells were selected by puromycin (2 μg/mL) and were maintained in EMEM media containing 10% FBS and 1 μg/mL puromycin. All cells were incubated at 37 ºC in a humidified atmosphere containing 5% CO 2 in a cell culture incubator.
8
Transient Transfection of N2a Cells
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Mouse neuroblastoma cells (n2a) from (ATCC ® CCL-131™), mycoplasma tested were transiently transfected using TransIT-Neural (Mirus) or FuGENE HD transfection reagent (Promega) according to manufacturer's protocol. 36 h post transfection, cells were harvested and lysed in TNG-T lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 % glycerol, 1 % Triton X-100, and proteinase inhibitor).
9
Culturing Neuro-2a Neuroblastoma Cells
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Neuro-2a neuroblastoma cells (ATCC® CCL-131™) were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultivated in DMEM medium (BioloT, St. Petersburg, Russia) supplemented with 10% of fetal bovine serum (BioloT, St. Petersburg, Russia) and 1% penicillin/streptomycin (BioloT, St. Petersburg, Russia) at 37 °C and 5% CO2. Initially, cells were incubated in cultural flasks until sub-confluent (~80%). For testing, Neuro-2a cells were seeded in 96-well plates and experiments were started after 24 h.
10
Neuro-2a Neuroblastoma Cell Culture
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Neuro-2a neuroblastoma cells (ATCC® CCL-131™) were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultivated in DMEM medium (BioloT, St. Petersburg, Russia) supplemented with 10% of fetal bovine serum (BioloT, Russia) and 1% penicillin/streptomycin (BioloT, St. Petersburg, Russia) at 37 °C and 5% CO2. Initially, cells were incubated in cultural flasks until sub-confluent (~80%). For testing, Neuro-2a cells were seeded in 96-well plates and experiments were started after 24 h [37 (link)].
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