For this study, the New York City Board of Health (NYCBOH) strain of VACV was used to immunize mice and the Western Reserve (WR) strain of VACV was used for viral challenge. Both virus strains were grown in Hela S3 cells (#CCL-2.2, ATCC). Virus titer was quantified in Vero cells (#CCL-81, ATCC) and stored − 80 °C for future use. For some experiments, viruses were ultraviolet-inactivated, following a protocol published previously25 (link).
Hela s3 cells
Manufactured by American Type Culture Collection
Sourced in United States
The HeLa S3 cells are a cell line derived from human cervical cancer cells. They are a widely used research tool in cell biology and biomedical research. The HeLa S3 cells provide a consistent and renewable source of human cells for various experimental purposes.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Hacat cells, by Addexbio (2 mentions)
Vero cell, by American Type Culture Collection (1 mentions)
Kanamycin, by Merck Group (1 mentions)
E coli bl21 de3, by New England Biolabs (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Bovine calf serum (bcs), by American Type Culture Collection (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Bl21 de3, by New England Biolabs (1 mentions)
Nico21 de3, by New England Biolabs (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Lys c, by Fujifilm (1 mentions)
28 protocols using hela s3 cells
1
VACV Strains for Immunization and Challenge in Mice
2
Bacterial Expression and Cell Culture
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Cloning was performed in E. coli DH5α, protein production performed in E. coli BL21 (DE3) (New England Biolabs, Ipswich, MA, USA). Bacteria were grown in an LB-Miller medium supplemented with 30 µg/mL kanamycin (Sigma, St. Louis, MO, USA). HeLa-S3 cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Gaithersburg, MD, USA) and Jurkat E6-1 (ATCC) in Roswell Park Memorial Institute medium (RPMI, Gibco). Both media were supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco), and cells were cultured at 37 °C in a 5% CO2 humidified atmosphere and subcultured every 2–3 days.
3
HeLa S3 Cell Culture and Lysate Preparation
4
Preparation and Characterization of HeLa S100 Cytosolic Lysates
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HeLa S3 cells (ATCC)
were maintained in Dulbecco’s modified eagle medium (DMEM)
with 10% v/v bovine calf serum (HyClone) and maintained in a 37 °C,
5% CO2 environment. OPM-2 cells (a kind gift from Donald
McDonnell) were maintained in RPMI 1640 media supplemented with 12%
fetal bovine serum (HyClone), 21.8 mM glucose, 8.6 mM HEPES (pH 7.4),
and 1.0 mM sodium pyruvate. All media components were from Cellgro
unless otherwise noted. All reagents used in following assays were
from Sigma-Aldrich unless otherwise noted. HeLa S100 cytosolic lysates
were generated from cells based on the Dignam protocol as previously
described.12 (link) Isolated HeLa S100 cytosolic
lysates were quantified with a Nanodrop 2000 (Thermo Scientific),
aliquoted, and stored at −80 °C.
were maintained in Dulbecco’s modified eagle medium (DMEM)
with 10% v/v bovine calf serum (HyClone) and maintained in a 37 °C,
5% CO2 environment. OPM-2 cells (a kind gift from Donald
McDonnell) were maintained in RPMI 1640 media supplemented with 12%
fetal bovine serum (HyClone), 21.8 mM glucose, 8.6 mM HEPES (pH 7.4),
and 1.0 mM sodium pyruvate. All media components were from Cellgro
unless otherwise noted. All reagents used in following assays were
from Sigma-Aldrich unless otherwise noted. HeLa S100 cytosolic lysates
were generated from cells based on the Dignam protocol as previously
described.12 (link) Isolated HeLa S100 cytosolic
lysates were quantified with a Nanodrop 2000 (Thermo Scientific),
aliquoted, and stored at −80 °C.
5
Poliovirus Infection and Replication Assay
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All experiments were conducted in HeLa S3 cells (ATCC, CCL-2.2) cultured in DMEM/F12 (Invitrogen) containing 10% FCS, penicillin and streptomycin and at 37 °C. The Mahoney type 1 (WT) and Sabin type 1 strains of poliovirus were generated by electroporation of in vitro transcribed RNA into HeLa S3 cells18 (link). For viral infections, cells were incubated with the virus for 30 min at 37 °C, after which media was added and infection allowed to continue until all cells were dead. Subsequently, two freeze-thaw cycles were performed to release intracellular viruses, cellular debris were removed by low speed centrifugation, and virus production was assessed by plaque assay. Geldanamycin (GA; LC labs) was diluted in DMSO and used at the indicated concentration. Monoclonal poliovirus antibodies were generously provided by Dr. Morag Ferguson at NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire.
6
Cell-Free Protein Expression Using HeLa Lysate
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HeLa-based CFE system includes HeLa lysate, truncated GADD34, T7 RNA polymerase, mix 1 and mix 2 solutions. The HeLa lysate was prepared from spinner-cultured HeLa S3 cells (ATCC Inc.). The detailed preparation steps can be found in a previously published work (19 ). Briefly, HeLa S3 cells were cultured in suspension using minimal essential medium eagle medium (SMEM) with Joklik modification without calcium, at an initial concentration of 1–2 × 105 cells/ml. Cells were harvested and lysed when the concentration reached 7–8 × 105 cells/ml after 4-5 days of culture. The harvested HeLa lysate was aliquoted and stored at −80°C. GADD34 and T7 RNA polymerase were prepared with the stock concentration of 2.3 and 5 mM, respectively. Mix 1 is a buffer prepared with 27.6 mM Mg(OAc)2, 168 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (K-HEPES pH 7.5). Mix 2 is a solution which was prepared with the following reagents: 12.5 mM ATP, 8.36 mM GTP, 8.36 mM CTP, 8.36 mM UTP, 200 mM creatine phosphate, 7.8 mM K-HEPES (pH 7.5) and 0.6 mg/ml creatine kinase. The pT7-CFE-GFP plasmid was prepared by cloning GFP protein into pT7-CFE-CHis vector (Thermo Fisher Scientific). The inserted GFP sequence can be found in Supplementary Table S2 .
7
Bacterial Cloning and Mammalian Cell Culture
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E. coli DH5α strain was used for cloning, E. coli BL21(DE3) or NiCo21(DE3) were used for protein expression (New England Biolabs). Bacteria were grown in LB-Miller medium, supplemented with antibiotic (ampicillin 100 μg/mL or kanamycin 30 μg/mL) when necessary. HeLa cells (HeLa-S3 cells, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotics (penicillin/streptomycin), at 37°C in a humidified atmosphere containing 5% CO2, and subcultured approximately every 2–3 days. All chemicals and reagents were purchased from Sigma.
8
Plating HeLa S3 Cells for Live Imaging
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HeLa S3 cells were purchased from ATCC and cultured in DMEM containing 10% fetal bovine serum in a humidified 5% CO2. Passage of HeLa S3 cells was performed every 3 or 4 days and plated HeLa S3 cells at the 1 to 10 ratio. To plate cells onto a coverglass Lab-Tek 8 well chamber, 50 μl of HeLa S3 suspension containing 3500 cells were placed at the center of each well and left until cells attached to the coverglass surface. Then, 0.75 ml of culture medium was added to each well. If a 0.75 ml cell suspension, instead of 50 μl, was added to the well, cells tended to be concentrated at the wall. Thus, our method gave a better result to evenly spread cells in a given area, although some local variation of cell concentration was still observed. Cells were used for live cell imaging 12 hrs after the plating.
9
Isolation and Culture of Primary OCP Cells
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HEK293T cells (ATCC) originating from a human fetal kidney and HeLa S3 cells (ATCC) originating from the female cervix were cultured in DMEM (Corning) supplemented with 10% FBS (Merck) and 1% penicillin-streptomycin (Corning).
To isolate primary OCP cells, bone marrow cells were collected from femurs and tibias of 6–8-week-old C57BL/6 mice and cultured in α-minimum essential medium supplemented with 10% FBS and M-CSF (5 ng·mL−1) for 16 h. Nonadherent cells were harvested and further cultured with M-CSF (30 ng·mL−1) for 3 days. After removal of floating cells, adherent cells were used as BMM cells.
To isolate primary OCP cells, bone marrow cells were collected from femurs and tibias of 6–8-week-old C57BL/6 mice and cultured in α-minimum essential medium supplemented with 10% FBS and M-CSF (5 ng·mL−1) for 16 h. Nonadherent cells were harvested and further cultured with M-CSF (30 ng·mL−1) for 3 days. After removal of floating cells, adherent cells were used as BMM cells.
10
In-StageTip Proteome Sample Preparation
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The complex proteome mixture used in our experiments was composed of HeLa S3 cells (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies Ltd) supplemented with 20 mM glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin. The sample preparation was performed following the in-StageTip protocol (20 (link)). After washing the cells in PBS and cell lysis, the proteins were reduced, alkylated, and digested by trypsin (Sigma-Aldrich) and LysC (WAKO) (1:100, enzyme/protein, w/w) in one step. The peptides were dried, resuspended in 0.1% TFA/2% acetonitrile (ACN), and 200 ng digest was loaded onto Evotips (Evosep). The Evotips were prepared by activation with 1-propanol, washed with 0.1% formic acid (FA)/99.9% ACN, and equilibrated with 0.1% FA. After loading the samples, tips were washed once with 0.1% FA.
The simple proteome mixture was composed in equal amounts of the purified and predigested tryptic standard proteins enolase, phosphorylase b, ADH, and BSA. The peptides were reconstituted in 0.1% FA, and either 200 or 400 fmol (see above) were loaded onto Evotips.
The simple proteome mixture was composed in equal amounts of the purified and predigested tryptic standard proteins enolase, phosphorylase b, ADH, and BSA. The peptides were reconstituted in 0.1% FA, and either 200 or 400 fmol (see above) were loaded onto Evotips.
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