The murine BV2 microglial cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco’s Modified Essential Medium (DMEM), supplemented with 100 units/mL penicillin–streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (Grand Island, NY, USA) in a humidified atmosphere of 5% CO2 at 37 °C. Cell viability was assessed by MTT assay to define the cell toxicity of ICSB. Briefly, BV2 cells at a density of 1 × 105 per well were cultured in a 96-well plate for 24 h, treated with different concentrations of ICSB (25, 50, and 100 μM) or vehicle alone for 2 h and were then co-cultured with LPS (500 ng/mL) at 37 °C for an additional 20 h. Following incubation, the cell-free culture mediums (100 µL) were collected for NO production by the Griess assay, and cell viability was measured as previously described [12 (link)].
Bv2 microglial cells
Manufactured by American Type Culture Collection
Sourced in United States
BV2 microglial cells are a commonly used murine microglial cell line derived from the opsoclonus myoclonus ataxia (OMA) mouse. They are immortalized microglial cells that have been utilized as an in vitro model to study various aspects of microglial biology and function.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Aβ1 42, by AnaSpec (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Cck 8 kit, by Merck Group (1 mentions)
P1400, by Solarbio (1 mentions)
Lipopolysaccharides (lps), by Solarbio (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by American Type Culture Collection (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Streptomycin sulfate, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Neuro 2a n2a, by American Type Culture Collection (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
18 protocols using bv2 microglial cells
1
Microglial Cell Viability Assay
2
Microglial Cell Activation and Inhibition Protocol
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BV‐2 microglial cells were purchased from ATCC and cultured with 45 mL DMEM complete medium (contained 5 mL 10% fetal calf serum). Cells were plated into 6well microtiter plates, treated with 20 μg/mL Aβ1‐42, which was purchased from Ana‐Spec (Fremont, CA, USA). Following 24 hours incubation, the cells were activated, which was as described as previously study (16). After 24 hours, microglia cultures were treated with an inflammatory inhibitor DHM at 2.5 μg/mL for 24 hours.17 , 18 Later cells were washed with phosphate‐buffered saline (PBS) and lysed in ice‐cold lysis buffer. Lysates were centrifuged at 15 000 g for 15 minutes at 4°C. The supernatants were collected and Samples were frozen at −80°C until further analysis.
3
BV2 Microglial Cells Stimulation
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BV2 microglial cells were purchased from ATCC (Manassas, VA, USA) and cultured according to the manufacturer's instructions. BV2 microglial cells were stimulated for 24 h with LPS (1 μg/ml) or vehicle control (HBSS) in DMEM high glucose media containing 2.5% FBS. Total RNA was isolated and identified as previously described.
4
Culture of Mouse BV-2 Microglia
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Mouse BV-2 microglial cells were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 0.1% penicillin-streptomycin (BioSource International, Camarillo, CA, United States) at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
5
Culturing BV2 and 293T Cells
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BV2 microglial cells (ATCC, #CRL-2467) were cultured in MEM supplemented with 10% FBS and 1% antibiotics. 293T cells (ATCC, #CRL-3216) were cultured in DMEM supplemented with 10% FBS and 1% antibiotics. All cells were routinely cultured in a humidified incubator in which 5% CO2 was supplied and maintained at 37°C.
Note: FBS used for culturing BV2 cells should be inactivated at 56°C for 30 min.
6
BV-2 Microglial Cells: LPS and 7,3',4'-THIF Treatments
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BV-2 microglial cells (catalog number: CRL-2469) were obtained from ATCC (Manassas, VA, USA). BV-2 microglial cells were maintained in DMEM supplemented with 10% heat-inactivated FBS (v/v) and 0.1% penicillin/streptomycin (v/v) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. When the cells reached 80-90% confluency in 100-mm2 cell culture dishes, they were dissociated with trypsin-EDTA and sub-cultured in culture dishes. LPS was prepared immediately before use as a 100 μg/ml stock and diluted in PBS to the indicated final concentration. 7,3’,4’-THIF was dissolved in DMSO and the stock solutions were added directly to the culture medium. In all experiments, cells were treated with the indicated concentrations of 7,3’,4’-THIF in serum-free DMEM with or without LPS (100 ng/mL) and control cells were treated with DMSO alone. The final concentration of solvent was always <0.1% (v/v).
7
Viability Assay of BV-2 Microglial Cells
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BV-2 microglial cells (ATCC, Rockville, MD, USA) were grown in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Frederick, MD, USA). Cells were maintained at 37 °C with 5% CO2 in a humidified atmosphere. All test concentrations of compounds showed no significant toxicity. The cell viability was determined by MTT assay.
8
Cytotoxicity Screening of DHN Derivatives
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The toxicity of DHN derivatives (6a-n, 7a-c ) was screened using CCK-8 kits (Sigma-Aldrich, MO, USA). The synthesised compounds were initially dissolved in dimethylsulphoxide (DMSO). Experimental concentrations of DMSO were always 0.1% (v/v), and concentrations of DHNs were 10 µM.
BV2 microglial cells (ATCC, Manassas, VA, USA) were cultured in 96-well plates then treated with DHN derivatives for 24 h. Next, 10 µL of the CCK-8 solution were added and incubated for 2 h at 37 °C, then absorbance at 450 nm was measured using a microplate reader. The survival rate (%) of BV2 microglia treated with compounds (10 µM) was then calculated. Results are the average of three replicates and shown inTable 1 .
BV2 microglial cells (ATCC, Manassas, VA, USA) were cultured in 96-well plates then treated with DHN derivatives for 24 h. Next, 10 µL of the CCK-8 solution were added and incubated for 2 h at 37 °C, then absorbance at 450 nm was measured using a microplate reader. The survival rate (%) of BV2 microglia treated with compounds (10 µM) was then calculated. Results are the average of three replicates and shown in
9
Culturing RAW 264.7 and BV2 Cells
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RAW 264.7 macrophages were obtained from the Korean Cell Lines Bank (KCLB, Seoul, Korea). BV2 microglial cells were obtained from the ATCC (Manassas, VA). RAW 264.7 macrophages and BV2 microglial cells were cultured in DMEM containing 10% FBS and 1% Penicillin at 37 °C in a 5% CO2 atmosphere. Media was changed once a day in all experiments.
10
BV2 Microglial Cell Culture
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BV2 microglial cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States). Cells were cultured in Dulbecco’s modified Eagle’s high glucose medium (DMEM, Gibco, USA). Subsequently, 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (P1400, Solarbio, China) were added to the DMEM. The cells were cultured in 5% CO2 at 37°C in an incubator.
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