HEK293T and HeLa cells were purchased from ATCC and grown at 37°C in DMEM medium supplemented with 10% fetal bovine serum and Penicillin-Streptomycin. Aedes albopictus C6/36 cells (ATCC CRL-1660) were maintained in RPMI-1640 medium with 2% FBS at 28°C. Neural progenitor cells derived from human H9 ES cells were cultured at 37°C in DMEM/F12 media, supplemented with 0.5X N2, 0.5X B27, 20 ng/μl bFGF.
Aedes albopictus c6 36 cells
Manufactured by American Type Culture Collection
Sourced in United States
Aedes albopictus C6/36 cells are an insect cell line derived from the Asian tiger mosquito, Aedes albopictus. These cells are commonly used in research as a model system for the study of arboviruses, which are viruses transmitted by arthropod vectors such as mosquitoes.
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Lab products found in correlation
Vero cell, by American Type Culture Collection (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Bhk 21, by American Type Culture Collection (3 mentions)
Schneider s insect medium, by Thermo Fisher Scientific (2 mentions)
Sh sy5y cell, by American Type Culture Collection (2 mentions)
Balb c mice, by Charles River Laboratories (2 mentions)
Bhk 21 cells, by American Type Culture Collection (2 mentions)
Vero e6 cell, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
L 15 medium, by Merck Group (1 mentions)
Pen strep, by American Type Culture Collection (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Atcc ccl 81, by American Type Culture Collection (1 mentions)
Atcc ccl 10, by American Type Culture Collection (1 mentions)
27 protocols using aedes albopictus c6 36 cells
1
Cell Culture Protocols for Diverse Cell Lines
2
Rift Valley Fever Virus Amplification
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RVFV strain 35/74 was originally isolated from the liver of a sheep that died during a RVFV outbreak in the Free State province of South Africa in 197421 (link). The strain was previously passaged four times in suckling mouse brain and three times in BHK cells. The virus used for IV inoculation of sheep was prepared by a further amplification in BHK-21 cells (ATCC CCL-10) cultured in CO2-independent medium (CIM, Invitrogen), supplemented with 5% FBS (Bodinco) and 1% Pen/Strep (Invitrogen).
To prepare a virus-spiked blood meal for membrane feeding of mosquitoes, the virus was amplified in Aedes albopictus C6/36 cells (ATCC CRL-1660). To this end, C6/36 cells were inoculated with a multiplicity of infection of 0.005 and cultured at 28 °C in absence of CO2 in L-15 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 2% Tryptose Phosphate Broth (TPB) and 1% MEM nonessential amino acids solution (MEMneaa). At 4 days post infection, culture medium was harvested, cleared by slow-speed centrifugation and titrated using Vero-E6 cells (ATCC CRL-1586), grown in DMEM supplemented with GlutaMAX, 3% FBS, 1% Pen/Strep and 1% Fungizone (DMEM +) at 37 °C and 5% CO2. Titers were determined using the Spearman-Kärber algorithm22 (link),23 .
To prepare a virus-spiked blood meal for membrane feeding of mosquitoes, the virus was amplified in Aedes albopictus C6/36 cells (ATCC CRL-1660). To this end, C6/36 cells were inoculated with a multiplicity of infection of 0.005 and cultured at 28 °C in absence of CO2 in L-15 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 2% Tryptose Phosphate Broth (TPB) and 1% MEM nonessential amino acids solution (MEMneaa). At 4 days post infection, culture medium was harvested, cleared by slow-speed centrifugation and titrated using Vero-E6 cells (ATCC CRL-1586), grown in DMEM supplemented with GlutaMAX, 3% FBS, 1% Pen/Strep and 1% Fungizone (DMEM +) at 37 °C and 5% CO2. Titers were determined using the Spearman-Kärber algorithm22 (link),23 .
3
Zika Virus Propagation in Cell Culture
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hNPC (human neural progenitor cell) was purchased from Merck (NJ, USA), and hNPC cells were cultured according to the manufacturer's instructions. SNB-19 (human glioblastoma cell line), A549 and 293FT cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA), 2 mM L-glutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin (Invitrogen, Carlsbad, CA). Aedes albopictus C6/36 cells (ATCC CRL-1660) were maintained at 28°C with 5% CO2 in DMEM supplemented with 10% FBS. Zika virus strain ZG-01 (GenBank accession number KY379148.1) was isolated by our group in 2016 [21 (link), 22 (link)], and was propagated in C6/36 cells. Viral stocks were stored at -80°C and titrated on C6/36 cells.
4
Isolation and Purification of DENV3
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Vero cells (ATCC CRL-1586) were cultured in minimum essential medium with 5% fetal bovine serum and 1% penicillin–streptomycin solution at 37°C with 5% CO2. Aedes albopictus C6/36 cells (ATCC CRL-1660) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum at 28°C.
The Hawaii strain of DENV1, the H87 strain of DENV3, and the H241 strain of DENV4 were provided by the Guangdong Provincial CDC. The Tr1751 strain of DENV2 was isolated from a patient with dengue fever. Viruses were propagated in C6/36 cells and titered in Vero cells by plaque forming assay.
Purified DENV3 particles were harvested from DENV3-infected C6/36 cells and concentrated by 8% polyethylene glycol precipitation. Then, DENV3 particles were purified from clarified extracts by ultracentrifugation.
The Hawaii strain of DENV1, the H87 strain of DENV3, and the H241 strain of DENV4 were provided by the Guangdong Provincial CDC. The Tr1751 strain of DENV2 was isolated from a patient with dengue fever. Viruses were propagated in C6/36 cells and titered in Vero cells by plaque forming assay.
Purified DENV3 particles were harvested from DENV3-infected C6/36 cells and concentrated by 8% polyethylene glycol precipitation. Then, DENV3 particles were purified from clarified extracts by ultracentrifugation.
5
Dengue Virus 1 Propagation in C6/36 Cells
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Serum or plasma from DENV-1-positive samples were used as inoculum for Aedes albopictus C6/36 cells (ATCC CRL-1660). Cells were harvested at days 6–7 of infection, and infections confirmed by indirect immunofluorescence with the monoclonal antibody Anti-Dengue Virus-1 MAB10227 (EMD Millipore Corporation, Billerica, MA, USA). Viral stocks were obtained with less than 3 passages in cell culture. Viral titers were assessed in plaque assays and recorded as plaque forming units/mL (PFU/mL). Briefly, viral suspension stocks were serially 10-fold diluted in MEM Eagle medium with 2% fetal bovine serum (FBS). Two hundred microliters were inoculated on cell monolayers in a 24-well plate. After 1 h of virus adsorption at 37°C, cells were overlayed with 3% of carboxymethylcellulose–MEM medium containing 2% FBS. After incubation at 37°C for 7 days, they were stained with crystal violet-formaldehyde, dried at room temperature and plaques were counted.
6
Cell Culture and Virus Propagation
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African green monkey kidney cells (Vero, ATCC-CCL-81) and baby hamster kidney fibroblast cells (BHK-21, ATCC-CCL-10) were purchased from the American Type Culture Collection (ATCC) and cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented by 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 incubator at 37 °C. Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 incubator at 27 °C. The JEV P3 strain (GenBank: U47032.1) was stored in our laboratory and was propagated and titrated on BHK-21 cells. ZIKV-MR-766 strain (GenBank: AY632535.2) was kindly provided by Dr. Xiaowu Pang (College of Dentistry, Howard University, USA) and was propagated and titrated on Vero cells.
7
Cell Culture Conditions for Flavivirus Research
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BHK-21 cells (ATCC CCL-10), L929 cells (ATCC CCL-1), SH-SY5Y cells (ATCC CRL-2266), and Vero cells (ATCC CCL-81) were cultured in Dulbecco’s minimal essential medium (DMEM; Invitrogen). A549 cells (ATCC CCL-185) and Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured in RPMI 1640. Aedes aegypti cell line Aag2 (ATCC CCL-125) was cultured in Schneider’s insect medium (Gibco). All the media above were supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. BHK-21, Vero, L929, and A549 cells were grown at 37°C in 5% CO2, whereas the C6/36 and Aag2 cell lines were grown at 28°C. The live attenuated vaccine 17D (GenBank accession no. MN072725 ) was recovered from the infectious plasmid pYF-17D (33 (link)). The viral stock was prepared and titrated using the standard plaque-forming assay in BHK-21 cells and stored at –80°C for further use.
8
Propagation and titration of Japanese encephalitis virus
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Vero cells (ATCC CCL-81) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine sera and 100 U/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37 °C in 5% CO2. Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured in RPMI 1640 medium containing 10% FBS at 28 °C CO2-free incubator. The yeast cell (Pichiapastoris strain X33) and expression plasmid pPICZA were obtained from Invitrogen (Carlsbad, CA, USA).
The JEV SA 14-14-2 strain, isolated from live-attenuated JEV vaccine manufactured by Chengdu Institute of Biological Products Co. Ltd. (Sichuan, China), was conserved in IMCAS. The JEV were propagated in C6/36 cells, titrated by standard plaque-forming assay on Vero cells, and stored at −80 °C.
BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Immune-deficient A129 mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College. Pigs were provided from Zhangwu Zhengcheng Pig Breeding Co., Ltd. Animals were randomly allocated to groups. All animal studies were performed blinded.
The JEV SA 14-14-2 strain, isolated from live-attenuated JEV vaccine manufactured by Chengdu Institute of Biological Products Co. Ltd. (Sichuan, China), was conserved in IMCAS. The JEV were propagated in C6/36 cells, titrated by standard plaque-forming assay on Vero cells, and stored at −80 °C.
BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Immune-deficient A129 mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College. Pigs were provided from Zhangwu Zhengcheng Pig Breeding Co., Ltd. Animals were randomly allocated to groups. All animal studies were performed blinded.
9
Propagation and Titration of Dengue Virus Serotype 2
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Dengue virus serotype 2 (DENV-2) (IND/P23085/1960 strain, GenBank accession no. JQ922552.1 ) was used in this study. Briefly, the virus propagation was done by infecting 70 to 80% confluent Aedes albopictus C6/36 cells (CRL-1660; ATCC) in serum-free medium at an MOI of 0.2, and the cells were thereafter incubated in L-15 medium with 2% FBS for 5 days. The supernatant was then harvested and concentrated 10 times. The virus was aliquoted and stored at −80°C. The titer of the virus was determined by performing the focus formation assay on Vero cells, as described previously (41 (link)).
10
Cell Culture Conditions for Flavivirus Research
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BHK-21 cells (ATCC CCL-10), L929 cells (ATCC CCL-1), SH-SY5Y cells (ATCC CRL-2266), and Vero cells (ATCC CCL-81) were cultured in Dulbecco’s minimal essential medium (DMEM; Invitrogen). A549 cells (ATCC CCL-185) and Aedes albopictus C6/36 cells (ATCC CRL-1660) were cultured in RPMI 1640. Aedes aegypti cell line Aag2 (ATCC CCL-125) was cultured in Schneider’s insect medium (Gibco). All the media above were supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. BHK-21, Vero, L929, and A549 cells were grown at 37°C in 5% CO2, whereas the C6/36 and Aag2 cell lines were grown at 28°C. The live attenuated vaccine 17D (GenBank accession no. MN072725 ) was recovered from the infectious plasmid pYF-17D (33 (link)). The viral stock was prepared and titrated using the standard plaque-forming assay in BHK-21 cells and stored at –80°C for further use.
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