For lentivirus-mediated stable introduction of sfGFP-DARPin-LA6 and NLSSV40-sfCherry-NLSMyc, we followed the methods described previously (Liu et al., 2021 (link)). Briefly, pVSV-G (PT3343-5; Clontech) and psPAX2 (plasmid #12260; Addgene; http://n2t.net/addgene:12260 ; RRID:Addgene_12260; a gift from Didier Trono), together with the pCDH vector (pCDH-NLSSV40-sfCherry-NLSMyc-Blast or pCDH-sfGFP-DARPin-LA6-Hygro) in a 1:3:4 weight ratio of each plasmid was transfected into ∼80% confluent 293T cells (CRL-3216; ATCC) using Lipofectamine 3000 following the manufacturer’s instructions for lentivirus production. One day after the transfection, the medium was replaced with fresh medium, which was harvested at 48 h after transfection. For virus infection, MEFs, C2C12, BJ-5ta, and MCF10A cells were incubated with the virus-containing culture supernatants with 4 µg/ml polybrene (Nacalai Tesque) for 24 h. Infected cells were selected by incubation in medium containing 200 µg/ml hygromycin B Gold or 3 µg/ml blasticidin S (InvivoGen) for 4 d, except that C2C12 cells were selected with 20 µg/ml blasticidin S for 4 d.
Blasticidin s
Manufactured by InvivoGen
Sourced in United States, Japan, France
Blasticidin S is a broad-spectrum antibiotic that inhibits protein synthesis in eukaryotic cells. It is commonly used as a selection agent for identifying and maintaining cells that have been successfully transfected or transduced with a gene of interest.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by InvivoGen (11 mentions)
Hygromycin b, by InvivoGen (8 mentions)
Puromycin, by Merck Group (6 mentions)
Zeocin, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Zeocin, by InvivoGen (5 mentions)
Polybrene, by Nacalai Tesque (5 mentions)
Pspax2, by Addgene (3 mentions)
Hygromycin b, by Roche (3 mentions)
Zeocin, by Roche (3 mentions)
Penicillin streptomycin, by Lonza (3 mentions)
Polybrene, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Irdye 680 goat anti mouse igg, by LI COR (2 mentions)
Irdye 800cw goat anti rabbit, by LI COR (2 mentions)
Superose 6 10 300 gl column, by GE Healthcare (2 mentions)
Vivaflow tangential filtration cassettes, by Sartorius (2 mentions)
Streptactin superflow column, by GE Healthcare (2 mentions)
77 protocols using blasticidin s
1
Lentivirus-Mediated Stable Gene Expression
2
Transposon and Lentiviral Gene Transfer Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For transposon-mediated gene transfer, cells were transfected with the PiggyBac or Tol2 transposase expression vector (mPBase or pCAGGS-T2TP; Table S1) and the donor vectors using 293fectin (Thermo Fisher Scientific, Waltham, MA). One day after tge transfections, cells were selected by 10 μg/mL blasticidin S (InvivoGen, San Diego, CA) or 1.0 μg/mL puromycin (InvivoGen). For lentivirus-mediated gene transfer, HEK-293T cells were cotransfected with each pCSII vector, psPax2 (a gift from Didier Trono; Addgene plasmid # 12260), and pCMV-VSV-G-RSV-Rev (a gift from Dr. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan), by using polyethyleneimine "Max" MW 40,000 (Polyscience, Warrington, PA). Virus-containing media were collected 48 hours after transfection, filtered, and applied to target cells with 10 μg/mL polybrene (Nacalai Tesque). Two days after infection, the infected cells were selected with the following antibiotics: 200 μg/mL hygromycin (Wako, Osaka, Japan), 800 μg/mL G418 (InvivoGen), 10 μg/mL blasticidin S (InvivoGen), 1.0 μg/mL puromycin (InvivoGen), and 10 μg/mL zeocin (InvivoGen). Single-cell clones were isolated by the limiting dilution method.
3
Transposon and Lentiviral Gene Transfer Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For transposon-mediated gene transfer, cells were transfected with the PiggyBac or Tol2 transposase expression vector (mPBase or pCAGGS-T2TP; Table S1) and the donor vectors using 293fectin (Thermo Fisher Scientific, Waltham, MA). One day after tge transfections, cells were selected by 10 μg/mL blasticidin S (InvivoGen, San Diego, CA) or 1.0 μg/mL puromycin (InvivoGen). For lentivirus-mediated gene transfer, HEK-293T cells were cotransfected with each pCSII vector, psPax2 (a gift from Didier Trono; Addgene plasmid # 12260), and pCMV-VSV-G-RSV-Rev (a gift from Dr. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan), by using polyethyleneimine "Max" MW 40,000 (Polyscience, Warrington, PA). Virus-containing media were collected 48 hours after transfection, filtered, and applied to target cells with 10 μg/mL polybrene (Nacalai Tesque). Two days after infection, the infected cells were selected with the following antibiotics: 200 μg/mL hygromycin (Wako, Osaka, Japan), 800 μg/mL G418 (InvivoGen), 10 μg/mL blasticidin S (InvivoGen), 1.0 μg/mL puromycin (InvivoGen), and 10 μg/mL zeocin (InvivoGen). Single-cell clones were isolated by the limiting dilution method.
4
Neutrophil Oxidative Burst Assay
5
Generation of Inducible TBEV NS Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells (ATCC CRM-CCL-2) were grown at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (EU approved, South America origin), 0.1 U/ml penicillin, and 0.1 μg/ml streptomycin. The medium, serum, and antibiotics were purchased from Gibco (Thermo Fisher Scientific). Flp-In HeLa cells hosting a FRT site in the genome were a kind gift from Stephen S. Taylor (University of Manchester, Manchester, UK). For generation of the NSP-GFP Flp-In T-REx HeLa cell line (NSP-GFP cells), Flp-In HeLa cells were cotransfected with the plasmids pcDNA5/FRT/TO (harboring TBEV subgenomic DNA encoding NS1-2A-2B-3-4A-4B-eGFP) and pOG44 using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. The transfected cells were grown in DMEM supplemented with 10% FBS (Gibco), 100 μg/ml hygromycin B (Invivogen), and 5 μg/ml blasticidin S (Invivogen) for plasmid selection. For inducible expression of TBEV NS proteins in Flp-In HeLa cells, doxycycline hyclate (Sigma-Aldrich) was added to the culture medium under the conditions specified.
6
Immunoblotting Antibody Panel Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary and secondary antibodies were used for immunoblotting: anti‐mouse OPN rabbit polyclonal antibody (IB1397; Immuno‐Biological Laboratories, Gunma, Japan), anti‐α‐tubulin (DM1A) mouse mAb (Millipore, Billerica, MA, USA), IRDye 680 goat anti‐mouse IgG and IRDye 800CW goat anti‐rabbit (LI‐COR Biosciences, Lincoln, NE, USA). Anti‐CD44 (GTX15834) was purchased from GeneTex (Irvine, CA, USA). Anti‐CD51 (104108), anti‐CD61 (104310), anti‐CD29 (102209), and rat or hamster IgG were purchased from BioLegend (San Diego, CA, USA). Phycoerythrin‐labeled anti‐Gr‐1 and allophycocyanin‐labeled anti‐Ly6G (1A8) were purchased from BioLegend. Collagen type C was purchased from Nitta Gelatin (Osaka, Japan). Blasticidin S and puromycin were purchased from InvivoGen (San Diego, CA, USA). Geltrex and Qtracker 655 were purchased from Thermo Fisher Scientific. DNase I was purchased from Roche Diagnostics (Basel, Switzerland). PD0325901 was purchased from Wako (Osaka, Japan).
7
Cell Transfection and Selection
8
Stable Cell Line for MHV Spike Ectodomain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate a stable Drosophila S2 cell line expressing recombinant MHV spike ectodomain, we used Effectene (Qiagen) and 2 μg of the plasmid encoding the MHV S protein ectodomain. A second plasmid, encoding blasticidin S deaminase was cotransfected as dominant selectable marker. Stable MHV S ectodomain expressing cell lines were selected by addition of 10 μg ml−1 blasticidin S (Invivogen) to the culture medium 48 h after transfection.
For large-scale production of MHV S ectodomain the cells were cultured in spinner flasks and induced by 5 μM CdCl2 at a density of approximately 107 cells per ml. After a week at 28 °C, clarified cell supernatants were concentrated 40-fold using Vivaflow tangential filtration cassettes (Sartorius, 10-kDa cut-off) and adjusted to pH 8.0, before affinity purification using StrepTactin Superflow column (IBA) followed by gel filtration chromatography using Superose 6 10/300 GL column (GE Life Sciences) equilibrated in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl. The purified protein was quantified using absorption at 280 nm and concentrated to approximately 4 mg ml−1.
For large-scale production of MHV S ectodomain the cells were cultured in spinner flasks and induced by 5 μM CdCl2 at a density of approximately 107 cells per ml. After a week at 28 °C, clarified cell supernatants were concentrated 40-fold using Vivaflow tangential filtration cassettes (Sartorius, 10-kDa cut-off) and adjusted to pH 8.0, before affinity purification using StrepTactin Superflow column (IBA) followed by gel filtration chromatography using Superose 6 10/300 GL column (GE Life Sciences) equilibrated in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl. The purified protein was quantified using absorption at 280 nm and concentrated to approximately 4 mg ml−1.
9
Generation of ZC3H12B Inducible Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flp-In T-REx-293 cells were seeded in 12-well plates and the following day the cells were transfected using Lipofectamine 2000 with 180 ng of pcDNA5/FRT/TO-ZC3H12B or pcDNA5/FRT/TO-ZC3H12B-D196A vectors and 1.42 µg of the pOG44 plasmid (Invitrogen). Twenty-four hours later the cells were trypsinized and split into 100-mm cell culture dishes in selection medium [DMEM with 4.5 g/L d -glucose supplemented with 10% tetracycline-free fetal bovine serum, 15 µg/mL blasticidin S and 100 µg/mL hygromycin B (Invivogen)]. The cells were left until visible formation of colonies. The colonies were transferred into 96-well plates, propagated and examined for expression of V5 tagged ZC3H12B after 24 h of induction with 1 µg/mL of doxycyline (Sigma-Aldrich). Single clones with similar level of induction of the ZC3H12B wild-type and D196A mutation bearing proteins were selected and used in further experiments. Additionally the selected clones were checked for zeocin sensitivity to confirm proper integration.
10
Evaluating TLR Activation in Transfected HEK-293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected HEK-293 cell lines stably expressing human TLR1/2, TLR2, TLR2/6, or TLR2/CD14 genes were gifts from Dr. Yan Xiang (University of Texas, Health Science Center at San Antonio). The cells carrying an empty plasmid, TLR1/2 plasmids, or TLR2/6 plasmids were cultured in high glucose DMEM (Dulbecco's Modified Eagle's Medium; Invitrogen, Gaithersburg, MD) with 10% FBS, streptomycin (100 μg/ml), penicillin (100 U/ml), and blasticidin S (10 mg/ml; InvivoGen, San Diego, CA). The cells carrying TLR2/CD14 plasmids were cultured in in high glucose DMEM supplemented with 50 mg/ml Hygromycin B Gold (InvivoGen, San Diego, CA). After 24 h culture in a 96-well microplate with an initial concentration of 500 cells/μl, the cells were co-cultured with E. coli LPS (1 μg/ml) or lipoproteins (1 to 0.001 μg/ml) for another 24 h. The lipoproteins were diluted in D10 medium (with 20 μg/ml gentamycin and with or without polymycin B 50 μg/ml) and incubated at 37°C for 1 h. The culture supernatants were collected for the ELISA analysis of IL-8 or mMIP-2 levels (DuoSet ELISA; R&D Systems, Inc. Minneapolis, MN). Results were obtained from cultures performed in triplicates.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!