A CHO cell line stably expressing the human CD18 subunit (CHO-CD18) was established and used to develop additional mutants expressing human CD11a, CD11b, and CD11c (CHO-CD11a/b/c) for use in candidalysin binding experiments as described.55 (link) HBEC-5i cells (CRL-3245, ATCC, Manassas, VA) were cultured in complete DMEM/F12 medium. N2a cells (CCL-131, ATCC, Manassas, VA) were cultured in complete DMEM. BV-2 cells were obtained from ATCC, determined as mycoplasma and chlamydia free and maintained frozen in liquid nitrogen. Cells were thawed, expanded in DMEM medium, and expression of standard microglial surface markers (TREM2, CD68, and MCM5 by RT-qPCR) was confirmed.9 (link)
Bv2 cells
Manufactured by American Type Culture Collection
Sourced in United States
BV2 cells are an immortalized mouse microglial cell line derived from the olfactory bulbs of neonatal C57BL/6 mice. These cells exhibit morphological and functional characteristics of microglia and are commonly used as a model for studying microglial biology and neuroinflammatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Control sirna, by Cell Signaling Technology (3 mentions)
Erk2 sirna, by Cell Signaling Technology (3 mentions)
Phage braf v600e plasmid, by Addgene (3 mentions)
Phage braf wt plasmid, by Addgene (3 mentions)
Gag pol retroviral plasmid, by Addgene (3 mentions)
Erk1 sirna, by Cell Signaling Technology (3 mentions)
Pbabe puro braf v600e plasmid, by Addgene (3 mentions)
Sapk jnk sirna, by Cell Signaling Technology (3 mentions)
Pspax2, by Addgene (3 mentions)
Pmd2 g, by Addgene (3 mentions)
High glucose dmem f12, by Thermo Fisher Scientific (3 mentions)
Raw 264.7 cells, by American Type Culture Collection (2 mentions)
High glucose dmem, by Thermo Fisher Scientific (2 mentions)
Sh sy5y cell, by American Type Culture Collection (2 mentions)
Clonidine, by Merck Group (1 mentions)
Propofol, by Merck Group (1 mentions)
39 protocols using bv2 cells
1
Engineered CHO Cell Lines for Candidalysin Binding
2
Hypoxia-Induced Microglial Cell Stress
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The BV2 cells (mouse microglial cells) were purchased from ATCC Company (Milan, Italy). The cells were suspended in DMEM (Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 U/mL of streptomycin) culture medium. At 80% confluency, the cells were treated with trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA), and the resulting cell suspension was adjusted to a cell density of 5 × 104 cells/mL. The cell suspensions were washed with PBS (Phosphate-Buffered Saline), resuspended in serum-free DMEM medium, and placed in an incubator at 37 °C, without and with the addition of Propofol, Dexmedetomidine, or Clonidine (Sigma–Aldrich, Milan, Italy). The drugs were tested at final concentrations of 25 and 50 μM. Hypoxia was then induced by challenging the cell cultures with a gas mixture containing 1.0% O2, 5% CO2, and 37 °C for 3 h to initiate hypoxia, followed by 3 h of reoxygenation at 37 °C using a gas mixture containing 5% CO2 and 18.0% O2. We used gas-controlled incubators to control the O2 levels of the cell cultures. In hypoxia, the O2 levels were 1%, and the CO2 levels were 5%. Under the reoxygenation conditions, the O2 levels were 18%, and the CO2 levels were 5%. Nitrogen was added to the incubator in order until the set O2% was achieved.
3
Modulation of Immune Cell Responses
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RAW264.7 cells, BV2 cells, and Vero cells were bought from ATCC. The genotypes of all the cell lines were verified, and the cells were mycoplasma free. RAW264.7 cells and BV2 cells were maintained in RPMI 1640 medium with 10% newborn calf serum and 100 U/ml penicillin-streptomycin (P/S) antibiotics in a 5% CO2 incubator. Vero cells were cultured in a medium (DMEM) supplemented with P/S and 10% newborn calf serum at 37°C with 5% CO2. Rapamycin and 3-MA were bought from Selleck. Before ZIKV infection, cells were treated with Rapamycin for 12 hours and 3-MA for 3 hours, respectively. SiRNA knockdown and CRISPR knockout were performed following the manufacturer's instructions (ATG7 siRNA, sc-41448, ATG5 siRNA, sc-41446, IFNAR1 siRNA, sc-40090, IFNAR2 sc-40092, sc-40092, IFNGR1 CRISPR Plasmids, sc-401191, IFNGR2 siRNA, sc-35635, IL10R2 siRNA, sc-75332, IFNLR1 siRNA, and sc-62498).
4
Microglial Cell Hypoxia-Reoxygenation Model
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Mouse microglial BV-2 cells were obtained from ATCC (Rockville, USA) and cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2 in a humidified incubator. For OGD/R treatment, after 80% confluence, the cells were switched from a normal feeding medium containing serum and glucose to an oxygen-glucose-deprived medium, and incubated in a hypoxic chamber with CO2/N2 (5%/95%) for 3 h. The cells were then returned to the normal feeding medium and incubated under normal conditions for different hours as reperfusion.
5
Microglial-Endothelial Cell Interactions
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BV-2 cells (a microglial cell line) were purchased from the ATCC. BV-2 cells were cultured in DMEM and supplemented with 10% FBS and 1% penicillin/streptomycin. BV-2 cells were treated with different concentrations of PDGF-BB (20 and 50 ng/mL) for 3 days. The culture medium was collected and centrifuged at 6000 rpm for 5 min. Then, the supernatant was collected and filtered by Millipak® 0.22 μm filter. Next, the CM was stored at -80℃ for further study.
The human umbilical vein endothelial cell lines (HUVECs) was purchased from ATCC and cultured in 6 wells plate with DMEM, 2% FBS, and 1% penicillin/streptomycin. HUVECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or with 1 mL of CM + 1 mL of growth medium for 1 d and harvested for further analysis.
Human brain microvascular endothelial cells (HBMECs) were obtained from Dr. Tianshi Que and purchased from Cell Systems (Seattle, WA, USA). They were cultured in 6-well plates in complete classical medium (Cell Systems) supplemented with 10% serum and culture boost (Cell Systems). HBMECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or combined with ALPL inhibitor (SBI-425, 50 µM, HY-124,756, MCE) for 1 d and harvested for further analysis.
The human umbilical vein endothelial cell lines (HUVECs) was purchased from ATCC and cultured in 6 wells plate with DMEM, 2% FBS, and 1% penicillin/streptomycin. HUVECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or with 1 mL of CM + 1 mL of growth medium for 1 d and harvested for further analysis.
Human brain microvascular endothelial cells (HBMECs) were obtained from Dr. Tianshi Que and purchased from Cell Systems (Seattle, WA, USA). They were cultured in 6-well plates in complete classical medium (Cell Systems) supplemented with 10% serum and culture boost (Cell Systems). HBMECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or combined with ALPL inhibitor (SBI-425, 50 µM, HY-124,756, MCE) for 1 d and harvested for further analysis.
6
Culturing Immortalized Murine Microglia
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BV2 cells (ATCC, UK), an immortalised murine microglial cell line, were cultured in RPMI-1640 medium (Sigma-Aldrich, UK) supplemented with 10% FBS (Gibco, UK) and 1% penicillin/streptomycin (Sigma-Aldrich, UK) until 70–80% confluent. Cells were detached with trypsin–EDTA (Sigma-Aldrich, UK), counted and seeded at a density of 13,000 cells/cm2.
7
Cortical Neuronal Cultures from BALB/c Mice
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Cortical neuronal cultures were prepared from BALB/c mouse pups, as previously described (42 (link)) with some modifications. Briefly, postnatal day 0 newborn pup brains were harvested for neuronal cultures. Cortical gray matter was dissected, plated separately in six-well plates at 1 × 106 per well (previously coated with laminin and poly-l -Ornithine, Sigma, St. Louis, MO, USA). Cultures were fed with neurobasal medium with B-27 supplements (Thermofisher, Waltham, MA, USA) and incubated at 37°C with 5% CO2. Seven-day-old neuronal cultures were treated for 16 h with 0.5 ml per well of microglial conditioned medium. Microglial conditioned media was obtained by incubating mouse microglia BV2 cells (ATCC CRL-2469) with LPS (10 µg/ml) in a humidified incubator (5% CO2, 37°C and 95% air) for 24 h.
8
BV2 Cell Culture and BRAF Transduction
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BV2 cells (ATCC, Manassas, VA, USA, Cat# CRL-2467, RRID: CVCL_5744) were cultured with high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and high glucose DMEM/F12 (Thermo Fisher Scientific) containing 10% FBS at 37°C in 5% CO2 under constant temperature and humidity. After transduction with BRAFV600E, BRAFWT, and vector lentivirus, respectively, for 24 hours, cells were cultured for 24 hours in DMEM/F12 without fetal bovine serum (FBS), and then medium and cells were collected for subsequent experiments. Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE-BRAFV600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE-BRAFWT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro-BRAFV600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).
9
Cytotoxicity Evaluation of NNFE in RAW 264.7 and BV2 Cells
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RAW 264.7 cells and BV2 cells (ATCC, Rockville, MD, USA) were cultured at 37 °C in DMEM supplemented with 10% FBS, streptomycin-penicillin (100 µg/mL each; Hyclone, South Logan, UT, USA) in a humidified atmosphere of 5% CO2. An adequate (5 × 105 cells/mL) number of cells were cultured in 96-well plates for 24 h, and then treated with predetermined concentrations of NNFE followed by 24-h incubation; MTT reagent was added to each well and further incubated at 37 °C for 1 h. Finally, 100% DMSO was used to dissolve the intracellular insoluble formazan and the absorbance was measured at 570 nm using a microplate reader (Victor3, PerkinElmer, Waltham, MA, USA) and the percentage viability was calculated [17 (link)].
10
Cell Culture Protocols for BV2 and SH-SY5Y
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The BV2 cells that were used in this study were purchased from ATCC (USA). They were cultured in Dulbecco's Modified Eagle Medium (DMEM) with the addition of 10% fetal bovine serum (FBS; Gibco, MA) and 100 μg/ml penicillin–streptomycin (Gibco, MA) in 5% CO2 at 37°C.
SH‐SY5Y human nerve cells were purchased from ATCC (USA) and were cultured in a 1:1 mixture of DMEM and F12 medium (Gibco, MA) with the addition of 10% FBS (Gibco, MA) in 5% CO2 at 37°C.
SH‐SY5Y human nerve cells were purchased from ATCC (USA) and were cultured in a 1:1 mixture of DMEM and F12 medium (Gibco, MA) with the addition of 10% FBS (Gibco, MA) in 5% CO2 at 37°C.
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