Sera (starting at a dilution of 1:4) were serially diluted in 96-well culture plates in DMEM supplemented with 2% FCS, P/S and Q. A tissue culture infectious dose (TCID50) of 100 units of SARS-CoV-2 (German isolate BavPat1/2020; European Virus Archive Global # 026 V-03883) was added to the serum dilutions in an equal volume of DMEM 2% FCS, P/S and Q. After incubation at 37 °C for 1 h, approximately 10,000 Vero C1008 cells (ATCC CRL-1586) were added to each well. Plates were then incubated at 37 °C with 5% CO2, and cytopathic effects (CPE) were evaluated at day 4 post infection. Neutralization was defined as complete reduction of CPE in serum dilutions compared to positive controls. Neutralization titers of three replicates were calculated as geometric means (reciprocal value). The lower detection limit of the assay is 8 and is determined by the first dilution of the respective serum including the added virus. Neutralization assays were performed in the BSL-4 laboratory of the Institute of Virology, Philipps-University Marburg, Germany.
Vero c1008 cells
Manufactured by American Type Culture Collection
Sourced in United States
Vero C1008 cells are adherent African green monkey kidney epithelial cells. They are used for the propagation and titration of various viruses.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Avicel rc 591, by DuPont (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Llc pk1 cells, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
U937 cells, by American Type Culture Collection (1 mentions)
Expicho s cells, by Thermo Fisher Scientific (1 mentions)
Expicho expression medium, by Thermo Fisher Scientific (1 mentions)
C6 36 cells, by American Type Culture Collection (1 mentions)
K562 cell, by American Type Culture Collection (1 mentions)
16 protocols using vero c1008 cells
1
SARS-CoV-2 Neutralization Assay
2
Viral Titration by Plaque Assay
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Viral titre was determined by plaque assay on Vero-C1008 cells (ATCC). Cells were plated at 650,000 cells per well and infected with 3 or 6 dilutions from a tenfold dilution series in 1% FBS/1× non-essential amino acids/MEM for 60 min at 37 °C, with rocking every 15 min. Overlay medium (2 ml) consisting of a 1:1 mix of 2× modified Eagle medium (Temin’s Modification) supplemented with 2 penicillin–streptomycin, 2× GlutaMAX, 10% FBS:2× Avicel RC-591 (Dupont) was added to cells. Cells were incubated for 3 days at 37 °C before the overlay medium was removed and cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. The fixative was removed, and cells were stained for at least 5 min with crystal violet (0.2% crystal violet (Sigma-Aldrich), 20% ethanol), before removal and plaque enumeration.
3
Cell Culture Conditions for Multiple Cell Lines
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The Vero C1008 cells (ATCC No. CRL-1586), the HEK293 cells (ATCC No. CRL-1573), and the Intestinal porcine epithelial cell line-J2 (IPEC-J2) cells (ACC701; Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured in the Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, MA, USA) and antibiotic-antimycotic (Gibco, Thermo Fisher Scientific, MA, USA). The HEK293F cells (Invitrogen, CA, USA) were cultured in FreeStyle™ 293 Expression Medium (Gibco, Thermo Fisher Scientific, MA, USA).
4
Propagation of PDCoV and PEDV in Cell Lines
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PDCoV isolates NT1_1215 (accession number KX361345) and PEDV isolate P1915-NPF-071511A (accession number KX981900) were used in the study. These two viruses were isolated from two pig herds experiencing PDCoV and PEDV outbreaks.
LLC-PK1 cells (ATCC CL-101) and Vero C1008 cells (ATCC CRL-1586) were used to propagate PDCoV and PEDV, respectively. Vero C1008 and LLC-PK1 cells were maintained using growth medium (Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA) for virus propagation. At 80% confluency, growth medium was discarded, and the cells were washed twice with 1X PBS (1X phosphate-buffered saline; 0.1 M, pH 7.2) followed by maintenance medium (DMEM (Gibco, USA) supplemented with 8 µg/ml trypsin/EDTA (Gibco, USA)). Each virus was added into each cell line and incubated at 37 °C with 5% CO2 for 60 min. After incubation, the inoculated cells were washed twice with 1X PBS. A maintenance medium was added to the inoculated cells, and the cells were incubated at 37 °C with 5% CO2 until a cytopathic effect (CPE) was observed.
LLC-PK1 cells (ATCC CL-101) and Vero C1008 cells (ATCC CRL-1586) were used to propagate PDCoV and PEDV, respectively. Vero C1008 and LLC-PK1 cells were maintained using growth medium (Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA) for virus propagation. At 80% confluency, growth medium was discarded, and the cells were washed twice with 1X PBS (1X phosphate-buffered saline; 0.1 M, pH 7.2) followed by maintenance medium (DMEM (Gibco, USA) supplemented with 8 µg/ml trypsin/EDTA (Gibco, USA)). Each virus was added into each cell line and incubated at 37 °C with 5% CO2 for 60 min. After incubation, the inoculated cells were washed twice with 1X PBS. A maintenance medium was added to the inoculated cells, and the cells were incubated at 37 °C with 5% CO2 until a cytopathic effect (CPE) was observed.
5
Propagation of Recombinant and Chimeric PEDV Viruses
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Vero C1008 cells (ATCC No. CRL-1586) were maintained in growth medium containing Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine protein (FBS), 250 ng/mL Amphotericin B, 100 U/mL Penicillin and 100 μg/mL Streptomycin. The recombinant viruses (iPEDVPT-P5 and iPEDVPT-P96), and the chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S), were propagated in post-inoculation medium (PI medium) containing DMEM supplemented with 0.3% tryptose phosphate broth (TBP), 0.02% yeast extract, and 10 μg/mL trypsin as described previously [18 (link)].
6
Cultivation and Infection of PEDV Viruses
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Vero C1008 cells (ATCC No. CRL-1586) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine protein (FBS), 250 ng/mL Amphotericin B, 100 U/mL Penicillin, and 100 μg/mL Streptomycin. Viral stock of PEDVPT-P96 in post-inoculation medium (PI medium) containing DMEM supplemented with 0.3% tryptose phosphate broth (TBP), 0.02% yeast extract (0.02%), and 10 μg/mL trypsin, as prepared in our previous study [15 (link)], was used for generating the infectious cDNA clone and as the control for in vivo and in vitro studies, whereas the virulent PEDVPT-P5 virus was used for animal challenge.
7
Culturing Diverse Cell Lines for Research
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ExpiCHO-S Cells (Cat# A29127; ThermoFisher Scientific, Waltham MA) were cultured in ExpiCHO Expression Medium (Cat# A2910001; ThermoFisher Scientific) and maintained at 37°C in 8% CO2 on a platform rotating at 125 rpm with a rotational diameter of 19 cm. They were subcultured according to the manufacturer’s instructions. HEK-293T/17 cells (Cat# CRL-11268, ATCC, Manassas, VA) and Vero-C1008 cells (Cat# CRL-1586, ATCC) were maintained in DMEM (Cat# 11965118; ThermoFisher Scientific) supplemented with 7% fetal bovine serum (FBS) (Cat# 26140079, lot 2358194RP, ThermoFisher Scientific) and 100 U/mL penicillin-streptomycin (Cat# 15140–122; ThermoFisher Scientific). Raji cells stably expressing DCSIGNR (Raji-DCSIGNR) [135 (link)] (provided by Ted Pierson, NIH), K562 cells (Cat# CCL-243, ATCC), and U937 cells (Cat# CRL-1593.2, ATCC) were maintained in RPMI 1640 supplemented with GlutaMAX (Cat# 72400–047; ThermoFisher Scientific), 7% FBS and 100 U/mL penicillin-streptomycin. C6/36 cells (Cat# CRL-1660, ATCC) were maintained in EMEM (Cat# 30–2003, ATCC) supplemented with 10% FBS at 30°C in 5% CO2. All cell lines were maintained at 37°C in 5% CO2 unless otherwise stated.
8
SARS-CoV-2 Serum Neutralization Assay
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The serum neutralization capacity against SARS-CoV-2 was assessed by VNT100 (virus neutralization test) as described previously53 (link). Briefly, vaccinee serum samples were heat-inactivated for 30 min at 56 °C and diluted in a two-fold dilution series (1:4–1:512) in 96-well cell culture plates, followed by addition of 100 plaque-forming units (PFU) of SARS-CoV-2 (German isolate BavPat1/2020; European Virus Archive Global #026 V-03883 (Genbank: MZ558051.1)). After 1 h of incubation at 37 °C, 2 × 104 Vero C1008 cells (ATCC, Cat. no. CRL-1586, RRID: CVCL_0574) were added. Cytopathic effects were evaluated at day 4 post infection. Neutralization was defined as the absence of cytopathic effects, and the reciprocal neutralization titer was calculated from the highest serum dilution without cytopathic effects as a geometric mean based on three replicates. The lower limit of detection (LLOD) is a reciprocal titer of 8, corresponding to the first dilution of the respective serum.
9
EBOV Neutralization Assay Protocol
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EBOV neutralization assay was performed as described by Ehrhardt et al.65 (link) Briefly, mouse sera were serially diluted and incubated with 100 TCID50 units of EBOV Mayinga (GenBank: NC_002549 ). Following incubation at 37°C for 1 h, Vero C1008 cells (ATCC CRL-1586) were added. Cytopathic effects were evaluated at day 7 p.i. Neutralization was defined as absence of CPE in serum dilutions. Neutralization titers of four replicates were calculated as geometric mean titers for sera (reciprocal value). The lower limit of detection (LLOD) of the assay is determined by the first dilution of the respective serum.
10
PEDV Pintung 52 Passage 7 Viral Stock
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The highly virulent viral stock of PEDV Pintung 52 passage 7 (PEDVPT-P7) was derived from PEDVPT-P5 (GenBank accession no. KY929405) as described in previous studies [19 (link),20 (link),21 (link)]. Vero C1008 cells (American Type Culture Collection no. CRL-1586) were used for viral preparation and the neutralizing assay. The culture medium was Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GE Healthcare, Uppsala, Sweden), 250 ng/mL amphotericin B, 100 U/mL penicillin, and 100 µg/mL streptomycin. The viral titer of PEDVPT-P7 was 1.78 × 105 TCID50/mL, as determined using the endpoint titration assay with a ten-fold serial dilution in triplicates.
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