Vero E6, Huh-7, 293T, and HeLa cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and Calu-3 2B4 cells (Kent Tseng, University of Texas Medical Branch) were grown in MEM supplemented with 20% FBS as previously described (41 (link)). Codon-optimized sMacro (nucleotides 3262 to 3783 of SARS-CoV MA15) and MERS-CoV ORF4a were synthesized and cloned into pUC57 (GenScript). The sMacro and GFP sequences were PCR amplified and ligated into a linearized pcDNA3 plasmid using In-Fusion (Invitrogen) cloning. The ORF4a sequence was PCR amplified and then restriction digested and ligated into the pLKO plasmid. The resulting constructs were confirmed by restriction digestion, PCR, and direct sequencing. Human IFN-α (B/D) and IFN-β were purchased from PBL (Piscataway, NJ). High-molecular-weight poly(I-C) and poly(dA-dT) were purchased from InvivoGen (San Diego, CA). Cells were transfected with either Polyjet (Amgen, Thousand Oaks, CA) or Lipofectamine 2000 (Fisher Scientific, Waltham, MA) per the instructions of the manufacturers.
Polyjet
Manufactured by Amgen
The Polyjet is a lab equipment product. It is a 3D printing technology used to create physical models and prototypes. The Polyjet system works by jetting photopolymer materials to build up parts in layers.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Calu 3 cells, by American Type Culture Collection (4 mentions)
Ifn γ, by R&D Systems (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Puc57, by GenScript (2 mentions)
Human ifn β, by R&D Systems (2 mentions)
High molecular weight poly 1 c, by InvivoGen (1 mentions)
Poly da dt, by InvivoGen (1 mentions)
Micropulser unit, by Bio-Rad (1 mentions)
M csf, by Merck Group (1 mentions)
Gm csf, by Merck Group (1 mentions)
Jetoptimus, by Polyplus Transfection (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Thincert, by Greiner (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Pneumacult ex plus medium, by STEMCELL (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Human ifn γ, by R&D Systems (1 mentions)
M csf, by GenScript (1 mentions)
6 protocols using polyjet
1
SARS-CoV-2 and MERS-CoV Protein Expression
2
Molecular Cloning and Expression of MERS-CoV Proteins
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E. coli DH10B (Gibco/BRL) cells were transformed by electroporation using a MicroPulser unit (Bio-Rad) according to the manufacturer’s instructions and grown on antibiotic-selective LB medium.
MERS-CoV ORF4a-HA and ORF4b-HA nucleotide sequences were synthesized and cloned into pUC57 (GenScript). They were then PCR amplified, restriction digested and ligated into the pLKO plasmid. A pLKO ORF4b-3XFLAG plasmid was made by creating a PCR product replacing the HA tag with a 3XFLAG tag. This PCR product was restriction digested and ligated back into the pLKO plasmid. The resulting constructs were confirmed by restriction digest, PCR, and direct sequencing. The sMacro and GFP pcDNA3 plasmids were previously described [55 (link)]. The NSP15-3XFLAG plasmid was a generous gift of Michael Buchmeier (University of California-Irvine), and the KPNA-FLAG plasmids were a generous gift of Megan Shaw (Mount Sinai Medical Center). Cells were transfected using Polyjet (Amgen) or Lipofectamine 2000 (Fisher Scientific) as per the manufacturer’s instructions.
MERS-CoV ORF4a-HA and ORF4b-HA nucleotide sequences were synthesized and cloned into pUC57 (GenScript). They were then PCR amplified, restriction digested and ligated into the pLKO plasmid. A pLKO ORF4b-3XFLAG plasmid was made by creating a PCR product replacing the HA tag with a 3XFLAG tag. This PCR product was restriction digested and ligated back into the pLKO plasmid. The resulting constructs were confirmed by restriction digest, PCR, and direct sequencing. The sMacro and GFP pcDNA3 plasmids were previously described [55 (link)]. The NSP15-3XFLAG plasmid was a generous gift of Michael Buchmeier (University of California-Irvine), and the KPNA-FLAG plasmids were a generous gift of Megan Shaw (Mount Sinai Medical Center). Cells were transfected using Polyjet (Amgen) or Lipofectamine 2000 (Fisher Scientific) as per the manufacturer’s instructions.
3
SARS-CoV-2 Cell Culture and Transfection
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Vero E6, Huh-7, Baby Hamster Kidney cells expressing the mouse virus receptor CEACAM1 (BHK-MVR) (gifts from Stanley Perlman, University of Iowa), and A549-ACE2 cells (a gift from Susan Weiss, University of Pennsylvania), were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Calu-3 cells (ATCC) were grown in MEM supplemented with 20% FBS. Human IFN-β and IFN-γ were purchased from R&D Systems. Cells were transfected with either Polyjet (Amgen) or Lipofectamine 3000 (Fisher Scientific) per the instructions of the manufacturers.
4
Diverse Cell Lines Cultured for Immunology Research
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NHDF, 293T, Hela-MVR, A549, A549-PARP14KO, A549-ACE2 (a generous gift from Susan Weiss, University of Pennsylvania), BHK, 17Cl-1, and Vero E6 cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Calu-3 cells (ATCC) were grown in MEM supplemented with 20% FBS. Baby hamster kidney cell line BSR-T7/5 constitutively expressing T7 RNA polymerase was maintained in Glasgow’s Minimum Essential Medium (G-MEM) with 10% FBS. Bone marrow derived cells were grown in RPMI supplemented with 10% FBS and differentiated into M0 macrophages using M-CSF (Millipore-Sigma) and dendritic cells (DCs) using GM-CSF (Millipore-Sigma). All cell lines were grown at 37 and 5% CO2. IFN-γ was purchased from R&D Systems and Poly(I:C) HMW (0.5μg/mL) was purchased from Millipore-Sigma. RBN012759 (PARP14i) was purchased from Atomwise. RBN012811 (DEG) and its associated negative control RBN013527 (CTL) were generously provided by Dr. Mario Niepel (Ribon Therapeutics). Cells were transfected with either Polyjet (Amgen), Lipofectamine 3000 (Fisher Scientific), or jetOPTIMUS (Polyplus) per the manufacturer’s instructions.
5
Culture and Expansion of Diverse Cell Lines
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Vero E6, Huh-7, Baby Hamster Kidney cells expressing the mouse virus receptor CEACAM1 (BHK-MVR) (gifts from Stanley Perlman, University of Iowa), and A549-ACE2 cells (a gift from Susan Weiss, University of Pennsylvania), were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Calu-3 cells (ATCC) were grown in MEM supplemented with 20% FBS. Human IFN-β and IFN-γ were purchased from R&D Systems. Cells were transfected with either Polyjet (Amgen) or Lipofectamine 3,000 (Fisher Scientific) per the instructions of the manufacturers.
Primary human bronchial epithelial cells (HBECs) were harvested from healthy donors whose lungs were rejected for transplant and with unknown airway disease. Passage 0 cells were expanded in PneumaCultTM-Ex Plus Medium (STEMCELL Technologies), supplemented with gentamicin (ThermoFisher Scientific) and amphotericin B (ThermoFisher Scientific). When the cells reached confluency, they were passaged and expanded a second time in bronchial epithelial cell growth medium before being seeded on translucent Thincert (Greiner), at a density of 2 × 105 cells in air-liquid interface media, as previously described (64 (link)) (SeeSI Appendix, Supplemental Methods for further details).
Primary human bronchial epithelial cells (HBECs) were harvested from healthy donors whose lungs were rejected for transplant and with unknown airway disease. Passage 0 cells were expanded in PneumaCultTM-Ex Plus Medium (STEMCELL Technologies), supplemented with gentamicin (ThermoFisher Scientific) and amphotericin B (ThermoFisher Scientific). When the cells reached confluency, they were passaged and expanded a second time in bronchial epithelial cell growth medium before being seeded on translucent Thincert (Greiner), at a density of 2 × 105 cells in air-liquid interface media, as previously described (64 (link)) (See
6
Establishment of SARS-CoV-2 Cell Lines and Macrophages
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Vero E6, Huh-7, Vero81, DBT, L929, HeLa cells expressing the MHV receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (HeLa-MHVR), Baby Hamster Kidney cells expressing the mouse virus receptor CEACAM1 (BHK-MVR) (all gifts from Stanley Perlman, University of Iowa), AJK6, and A549-ACE2 cells (both gifts from Susan Weiss, University of Pennsylvania), were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Calu-3 cells (ATCC) were grown in MEM supplemented with 20% FBS. Bone marrow-derived macrophages (BMDMs) sourced from PARP12+/+ and PARP12−/− mice were differentiated into M0 macrophages by incubating cells in Roswell Park Memorial Institute (RPMI) media supplemented with 10% FBS, sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin, L-glutamine, M-CSF (Genscript) for six days. Then to differentiate into M2 macrophages, IL-4 (Peprotech Inc.) was added for 1 day. Cells were washed and replaced with fresh media every other day after the 4th day. Human IFN-γ was purchased from R&D Systems. Cells were transfected with either Polyjet (Amgen) or Lipofectamine 3,000 (Fisher Scientific) per the instructions of the manufacturers.
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