We used the clinical isolates dengue ST (passage 6) [35 (link)] and Chikungunya EAS DMERI09/08 (passage 3) viruses, collected from the Singapore General Hospital in 1997 and National University Hospital, Singapore in 2008, respectively (already existing collection). A portion of the E1 gene from CHIKV EAS was amplified and sequenced to validate the absence of the A226V amino acid substitution [9 (link)]. All viruses were propagated in Vero cells (ATCC) and titrated by plaque assay in BHK cells (ATCC) as previously described [36 (link)].
Bhk cells
Manufactured by American Type Culture Collection
Sourced in United States
BHK cells are a type of cell line derived from baby hamster kidney cells. They are commonly used in cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Vero cell, by American Type Culture Collection (3 mentions)
Mcf 7 cell, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
L929 cell, by American Type Culture Collection (1 mentions)
Vero e6 cell, by American Type Culture Collection (1 mentions)
Anti his apc antibody, by Miltenyi Biotec (1 mentions)
Facscanto, by BD (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Pvax1, by Thermo Fisher Scientific (1 mentions)
Pet28a vector, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Puromycin, by Corning (1 mentions)
Hygromycin b, by Corning (1 mentions)
Hek293t cells, by Corning (1 mentions)
Penicillin streptomycin pen strep, by Corning (1 mentions)
Geneticin, by Corning (1 mentions)
293ft cells, by Corning (1 mentions)
Vero cells, by Corning (1 mentions)
9 protocols using bhk cells
1
Propagation and Validation of Dengue and Chikungunya Viruses
2
Propagation and Titration of Viruses
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The LCMV strain WE and VSV (strain Indiana) were kindly provided by Rolf Zinkernagel (Institute of Experimental Immunology, ETH, Zurich, Switzerland). LCMV was propagated in L929 cells, which were purchased from ATCC (CCL-1). VSV was propagated in BHK cells, which were bought from ATCC (CRL-8544). Virus titres in tissue of infected mice were measured using plaque assays. For detection of virus in the skin, about 10 mg of the infected skin area was used. Candid#1 was grown in Vero E6 cells purchased from ATCC (CRL-1586). Recombinant VSV-GP (provided by Professor von Laer, Division of Virology, Medical University Innsbruck, Austria) was generated as previously described38 (link). VSV-GP was grown on Vero cells under serum-free conditions. Recombinant Tk− Vaccinia virus strain Western Reserve containing the green fluorescent protein gene expressed under the P7.5 promoter within the Tk (thymidine kinase) locus of the genome (originally provided by B. Moss, National Institutes of Health, Bethesda, MD) was propagated, purified and titrated following standard methodology65 .
3
Quantifying SARS-CoV-2 Protein Binding to ACE2
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The ACE2‐pEGFP‐N1 plasmid was transfected into BHK cells (ATCC, CCL‐10) using polyethylenimine (Alfa). When cell fluorescence could be observed with a fluorescence microscope within 24–48 h, the cells were collected, washed twice with PBS, and distributed into 96‐well plates. The cells were incubated with test proteins (SARS‐CoV‐2 RBD, SARS‐CoV‐2 NTD, and RshSTT182/200 RBD with histidine tag) at a concentration of 30 μg/ml at 37°C for 30 min. Then, the cells were washed three times with PBS and stained with anti‐His/APC antibody (1:500, Miltenyi Biotec) at 37°C for 30 min. Finally, the cells were analyzed using a BD FACSCanto (BD Biosciences) after washing them three times with PBS. All data were analyzed using FlowJo V10.
4
Quantifying BC200 RNA in Breast Cancer
5
EHDV-2 Isolate Characterization Protocol
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The EHDV-2 isolate used for inoculation was originally isolated at the Southeastern Cooperative Wildlife Disease Study from the spleen of a free-ranging WTD (CC12-304) from Coffey County, Kansas, during a 2012 EHD outbreak. The virus was originally isolated on cattle pulmonary artery endothelial (CPAE) cells (American Type Culture Collection, Manassas, VA, USA), passaged once in baby hamster kidney (BHK) cells (ATCC), and then to CuVaW8A (CuVa) cells (Culicoides sonorensis cell line; USDA-ARS) [18 (link),19 (link)]. The virus stock was 106.2 tissue culture infective doses (TCID50)/mL as determined by virus titration using CPAE cells in a 96 well format as described in [13 (link)] and endpoint titers were determined [20 ]. Sham inoculum for negative control contained culture media from cell culture flasks not inoculated with virus.
6
Plasmid Expression Characterization
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Recombinant eukaryotic and prokaryotic plasmids were identified by restriction enzyme digestion and verified by sequencing analysis. The inserts were subcloned into either a pVAX1 or pET28a vector (Invitrogen, Carlsbad, CA, USA). Expression levels were examined by Western blot analysis 48 hours after transfection of BHK cells (American Type Culture Collection, Manassas, VA, USA) for inserts in the pVAX backbone or 8 hours after induction of transfected Escherichia coli with 0.1 mM isopropyl-β-D-thiogalactopyranoside for inserts in pET28 vectors.
7
Cloning and Mutagenesis of αIIbβ3 Integrin
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Complementary DNA (cDNA) of β3 and αIIb was cloned into pcDNA3 and pCEP4 vectors, respectively, as previously described.6 (link) C490A or C545A mutations in αIIb were made by using Site-directed Ligase-Independent Mutagenesis and confirmed by sequencing. Vectors were linearized by using AvrII for αIIb-pCEP4 and PVUI for β3-pcDNA3, then underwent isopropanol clean-up before transfection. BHK cells from American Type Culture Collection maintained in Dulbecco’s modified Eagle medium with 10% FBS, GlutaMAX, and 1% penicillin-streptomycin solution were cotransfected with either wild-type (WT) or C490A or C545A mutant αIIb-pCEP4 vector and WT β3-pcDNA3 vector using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Antibiotic selection of transfected cells was initiated 48 hours after transfection and maintained in culture. Cells were checked for αIIbβ3 expression by flow cytometry 1-week posttransfection and then sorted based on CD61 expression (clone VI-PL2, Thermo Fisher Scientific). At passaging, cells were washed in PBS and overlayed with 5 mM EDTA in PBS or TrypLE (Gibco) until detached. Cells were then resuspended in fresh media and seeded into a new flask. Cells were used for analysis between passages 3 and 20.
8
BHK Cell Culture and Transfection
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Naïve Baby Hamster Kidney (BHK) cells (purchased from American Type Culture Collection [ATCC]; Manassas, USA) were maintained in DMEM/F12 media containing 5% fetal bovine serum under standard culture conditions (37°C, 5%CO2). BHK cells stably expressing wild-type CFTR (CFTRwt) [17 (link)] or the ΔF508 CFTR mutant (CFTRΔF508) [17 (link)] were maintained in medium supplemented with 500μmol/L methotrexate (which activates the CFTR transgene promoter).
Cells (at 40–50% confluency) were transfected with plasmid DNA (2μg DNA per 35mm dish) or siRNA (25nmol/L per 22mm dish) using FuGene 6 transfection reagent (Promega; Madison, USA), according to the manufacturer’s instructions. Cells were transfected for 48 hours, at which point cell confluency was approximately 95%. This investigation utilized plasmids encoding mutated CFTR proteins containing either a serine-to-alanine mutation at residue 737 (CFTRS737A) or a glycine to aspartic acid mutation at residue 551 (CFTRG551D); siRNA reagents (“On-Target plus” siRNA targeting the human AMPK α1 catalytic subunit [PRKAA1; cat# L-005027–00] and control, non-targeting siRNA [cat# D-001810-10-05]) were purchased from Dharmacon, Inc. (Lafayette, USA).
Cells (at 40–50% confluency) were transfected with plasmid DNA (2μg DNA per 35mm dish) or siRNA (25nmol/L per 22mm dish) using FuGene 6 transfection reagent (Promega; Madison, USA), according to the manufacturer’s instructions. Cells were transfected for 48 hours, at which point cell confluency was approximately 95%. This investigation utilized plasmids encoding mutated CFTR proteins containing either a serine-to-alanine mutation at residue 737 (CFTRS737A) or a glycine to aspartic acid mutation at residue 551 (CFTRG551D); siRNA reagents (“On-Target plus” siRNA targeting the human AMPK α1 catalytic subunit [PRKAA1; cat# L-005027–00] and control, non-targeting siRNA [cat# D-001810-10-05]) were purchased from Dharmacon, Inc. (Lafayette, USA).
9
Cell Lines and Virus Propagation Protocol
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HEK293T cells, Vero cells, BHK cells and HUVEC were purchased from the American Type Culture Collection (ATCC). EA.hy926 cells were purchased from the tissue culture facility at the University of North Carolina at Chapel Hill. 293FT cells were purchased from Thermo Fisher. iSLK.219 cells were a kind gift from Don Ganem. HEK293T cells, Vero cells, BHK cells, 293FT cells, and EA.hy926 cells were maintained in Dulbecco’s minimal essential medium (DMEM) (Corning) containing 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin-streptomycin (Pen-Strep) (Corning). HUVEC were maintained in EGM-2 supplied with growth factors obtained from the EGM-2 Bullet kit (Lonza). iSLK.219 cells were maintained in DMEM containing 10% tetracycline (Tet)-free FBS (Sigma), 1% Pen-Strep, 10 μg/ml puromycin (Corning), 50 μg/ml Geneticin (Corning), and 100 μg/ml hygromycin B (Corning). All cells were maintained at 37°C in a 5% CO2 laboratory incubator which was subject to routine cleaning and decontamination. HSV-1 (KOS strain) was obtained from ATCC and propagated in Vero cells. VSV was a kind gift from Doug Lyles and was propagated in BHK cells. KSHV was purified form iSLK.219 cells in our lab.
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