Supernatants from transfected 293T cells were harvested at 36 and 60 h posttransfection, filtered through a 0.45-μm syringe filter, and used to transduce THP-1 cells in the presence of 2 μg/ml Polybrene. When necessary, lentiviral titers were determined by in-house p24 enzyme-linked immunosorbent assay (ELISA). For transduction with puromycin resistance vectors, puromycin (2 μg/ml, Sigma) was added to media and refreshed every 2 to 5 days until all control nontransduced THP-1 cells were dead. Similarly, where blasticidin resistance vectors were used, blasticidin (1 μg/ml; Invivogen) was added to media. Emerald-positive cells were sorted using a BD FACSAriaIII instrument.
Blasticidin
Manufactured by InvivoGen
Sourced in United States, France, United Kingdom, Canada, Germany, China, Hong Kong
Blasticidin is a selection antibiotic used for the generation of stable cell lines. It inhibits protein synthesis by preventing peptide bond formation.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by InvivoGen (113 mentions)
Zeocin, by InvivoGen (95 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (82 mentions)
Polybrene, by Merck Group (55 mentions)
Normocin, by InvivoGen (47 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (47 mentions)
Hygromycin, by InvivoGen (46 mentions)
Puromycin, by Merck Group (45 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (38 mentions)
Streptomycin, by Thermo Fisher Scientific (36 mentions)
Penicillin, by Thermo Fisher Scientific (34 mentions)
Doxycycline, by Merck Group (31 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (27 mentions)
Pspax2, by Addgene (23 mentions)
Glutamax, by Thermo Fisher Scientific (23 mentions)
Hygromycin b, by InvivoGen (22 mentions)
L glutamine, by Thermo Fisher Scientific (21 mentions)
Puromycin, by Thermo Fisher Scientific (20 mentions)
Zeocin, by Thermo Fisher Scientific (18 mentions)
Opti mem, by Thermo Fisher Scientific (16 mentions)
577 protocols using blasticidin
1
Lentiviral Transduction of THP-1 Cells
2
Genetic Manipulation of Multiple Myeloma
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Human multiple myeloma cell lines were transduced with virus containing with or without the CDK6 construct in backbones pLenti.6.2.V5-DEST and pRSF91-GFP-T2A-Puro. Transduced cells were selected with blasticidin and puromycin (InvivoGen, San Diego, USA) respectively.
Human multiple myeloma cell lines were transduced with virus containing pLKO5d.SSF.SpCas9.P2a.BSD and cells were selected with blasticidin (Invivogen, San Diego, USA). Cells were then transduced with respective CRBN and RB1 sgRNA constructs cloned into pLKO5.hU6.sgRNA.dTom, along with luciferase and POLR2A controls. Transduction success was confirmed through FACS analysis 48 h post-transduction with a minimum efficiency of 95% tomato fluorescence. CRBN and RB1 knockout was confirmed through Western blot analysis. CRBN-targeting sgRNAs are as follow: 5′-GTCCTGCTGATCTCCTTCGC-3′ and 5′- GGATTCACATAAGCTGCCAT-3′; RB1-targeting sgRNA is as follow: 5′- GGTTCTTTGAGCAACATGGG-3′.
Human multiple myeloma cell lines were transduced with virus containing pLKO5d.SSF.SpCas9.P2a.BSD and cells were selected with blasticidin (Invivogen, San Diego, USA). Cells were then transduced with respective CRBN and RB1 sgRNA constructs cloned into pLKO5.hU6.sgRNA.dTom, along with luciferase and POLR2A controls. Transduction success was confirmed through FACS analysis 48 h post-transduction with a minimum efficiency of 95% tomato fluorescence. CRBN and RB1 knockout was confirmed through Western blot analysis. CRBN-targeting sgRNAs are as follow: 5′-GTCCTGCTGATCTCCTTCGC-3′ and 5′- GGATTCACATAAGCTGCCAT-3′; RB1-targeting sgRNA is as follow: 5′- GGTTCTTTGAGCAACATGGG-3′.
3
Cell culture and virus strains protocol
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BSC-1 cells (ATCC CCL-26) were grown in Eagle’s Minimal Essential Medium (EMEM) supplemented with 0.1mg/ml penicillin, 0.1mg/ml streptomycin, 2 mM L-glutamine (BioWhittaker) and 5% fetal bovine serum (FBS). Flp-In™ 293 T-REx cells (Invitrogen-Life Technologies), derived from 293 human embryonic kidney cells, were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 0.1mg/ml penicillin, 0.1mg/ml streptomycin, 2 mM L-glutamine (BioWhittaker) and 7% fetal bovine serum (FBS). Cells were maintained in medium containing 5 μg/ml Blasticidin (Invivogen) and 300 μg/ml Zeocin (Invivogen). MxA-293T cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 0.1mg/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (BioWhittaker) and 7% fetal bovine serum (FBS) 5 μg/ml Blasticidin (Invivogen) and 100 μg/ml Hygromycin (Invivogen).
VSV-ΔG was kindly provided by Brian Lichty, McMaster University, Canada [11 (link)] [12 (link)]. VSV-GFP [13 (link)] was obtained from Sean P. J. Whelan, Harvard medical school USA. CPXV BR and EP4 were obtained from A. Alcamí (Centro de Biologia Molecular, Madrid).
VSV-ΔG was kindly provided by Brian Lichty, McMaster University, Canada [11 (link)] [12 (link)]. VSV-GFP [13 (link)] was obtained from Sean P. J. Whelan, Harvard medical school USA. CPXV BR and EP4 were obtained from A. Alcamí (Centro de Biologia Molecular, Madrid).
4
Murine Ovarian Cancer Cell Line ID8 Culture
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The murine ovarian cancer cell line ID8, a gift from Dr. Katherine Roby (University of Kansas Medical Center, Kansas City, KS)70 (link), was cultured in DMEM supplemented with 4% FBS and 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite (all Sigma-Aldrich). To generate the more aggressive vascular endothelial growth factor (VEGF)-expressing strain, we transfected ID8 tumor cells with the pUNO1 plasmid (Invivogen) encoding murine VEGF and the blasticidin-resistance gene. To obtain stable transfectants, tumor cells were cultured in complete medium containing 10 μg/ml blasticidin (InvivoGen) for three weeks. The THP1-LuciaTM ISG cells (interferon regulatory factor-inducible reporter monocytes) were purchased from InvivoGen (Cat# thp1-isg) and cultured in RPM1–1640 supplemented with 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum (FBS). All cell lines tested negative for mycoplasma based on DNA-based PCR tests (DDC Medical).
5
Engineered Tet-On Cell Lines for BTG3 Regulation
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To establish inducible osteosarcoma U2OS and prostate cancer PC3 Tet-On stable cell lines for BTG3 knockdown or overexpression, U2OS-TR cells (tetracycline regulator-expressing)46 (link) or PC3-TR cells were stably transfected with pBabe-H1-shBTG34 (link) or pcDNA4-TO-BTG3, respectively. The U2OS BTG-knockdown stable cell line was maintained regularly in complete DMEM containing 2.5 μg/ml blasticidin (InvivoGen, San Diego, CA, USA) and 0.8 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA), and the PC3 BTG3-overexpressed line in F-12 K plus 2.5 μg/ml blasticidin and 100 μg/ml Zeocin (InvivoGen). Induction was performed using 1 μg/ml doxycycline (Sigma-Aldrich).
The human embryonic kidney 293T cells and the prostate cancer cell line DU145 were maintained, respectively, in DMEM and RPMI containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco, Life Technologies, Grand Island, NY, USA).
The human embryonic kidney 293T cells and the prostate cancer cell line DU145 were maintained, respectively, in DMEM and RPMI containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco, Life Technologies, Grand Island, NY, USA).
6
Zika Virus ZIKV Infection Protocol
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The clinical isolate PF-25013-18 (PF13) of ZIKV has been previously described [10 (link)]. Cell lines used in this study included the A549-Dual™cell line (InvivoGen, a549d-nfis), referred to hereafter as “A549 cells”, and the HEK-Blue™ IFN-α/β cell line (InvivoGen, San Diego, CA, USA) which possesses the HEK-293A backbone and is referred to hereafter as “HEK-293A cells”. Both cell lines were cultured at 37 °C with 5% CO2 in MEM Eagle medium, supplemented with 10% heat inactivated fetal bovine serum and 2 mmol·L−1l -Glutamine, 1 mmol·L−1 sodium pyruvate, 100 U·mL−1 of penicillin, 0.1 mg·mL−1 of streptomycin and 0.5 µg·mL−1 of fungizone (PAN Biotech, Aidenbach, Germany). Additionally, A549 cellular growth medium was supplemented with 10 µg·mL−1 blasticidin and 100 μg·mL−1 zeocin (InvivoGen) and HEK-293A cell growth medium was supplemented with 30 µg·mL−1 blasticidin and 100 μg·mL−1 zeocin (InvivoGen). Cells were harvested and stored as frozen pellets for further protein or mRNA analysis.
7
Lentiviral Transduction of ABCD Knockout Cell Lines
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The open reading frames of the ABCD1, ABCD2, or ABCD3 genes were cloned into the lentiviral vector pLENTI 6.3/TO/V5-DEST (Invitrogen). Hek-293T cells were cotransfected with one of the generated plasmids and the lentiviral packaging plasmids pMD2G, pMDL/RRE, and pRSV/REV using jetPRIME (Polyplus) to produce lentiviruses. Virus-containing media were collected 48 and 72 h after transfection, filtered through 0.45 μm filters, and used to transduce ΔABCD1ΔABCD3 clone A, ΔABCD1ΔABCD3 clone B, or ΔABCD3ΔACOX1 cells. Blasticidin-resistant cells were selected with 3 μg/ml Blasticidin (InvivoGen) for 7–10 days. After selection, cells were allowed to recover for at least 1 week prior to experiments.
8
HeLa T-REx Cell Culture and Transfection
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HeLa T-REx (Invitrogen, Paisley, UK) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/GlutaMAX (Gibco, Paisley, UK). Transient transfection of HeLa T-REx cells was performed using TransIT®-LT1 (Mirus, Madison, USA) according to manufacturer’s instructions. Stable transfection of HeLa T-REx cells for the inducible expression of BCR-ABL, HA-EpoR, vSrc-dsRed and Hck-dsRed were generated with the Flp-In system (Invitrogen, Paisley, UK) using 250 μg/ml hygromycin B (PAA, Austria) and 15 μg/ml blasticidin (Invivogen, Toulouse, France) for selection. Non-inducible HeLa T-REx FRT cells were maintained in DMEM medium supplemented with 200 μg/ml Zeocin (Invivogen, Toulouse, France) and 15 μg/ml blasticidin. The expression of the gene of interest was induced with 5 ng/ml doxycycline (Sigma, St. Louis, USA) for 24 h or as indicated. All media were supplemented with 10% FCS (Lonza, Verviers, Belgium) and 25 U/ml penicillin/streptomycin (Lonza, Verviers, Belgium). Cells were incubated at 37°C in a water-saturated atmosphere with 5% CO2.
9
Lentiviral Transduction and Genetic Modification of 721.221 Cells
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Lentiviral transduction for each sequential transduction was performed as described (Garcia-Beltran et al., 2016 (link)). 721.221 cells were transduced with lentivirus encoding LentiCas9-Blast and then selected with 5 μg/mL blasticidin (Invivogen). 721.221 + Cas9 cells were subsequently transduced with lentivirus encoding HLA class I genes and selected in 0.5 μg/ml puromycin (Invivogen). High HLA class I expressing cells were subcloned by limiting dilution. High-expressing HLA class I clones were then transduced with lentivirus encoding pLenti-sgRNA targeting exon 3 of the human TAP1 gene, followed by selection in 1.5 mg/ml G418 (Invivogen). Cells with low HLA class I surface expression following were subcloned by limiting dilution. Cas9, HLA and TAP sgRNA-expressing 721.221 cells were maintained in 5 μg/mL blasticidin (Invivogen), 0.5 μg/ml puromycin (Invivogen) and 1.5 mg/ml G418 (Invivogen).
10
Engineered Cell Lines for SARS-CoV-2 Research
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2.5 × 105 HEK-293 cells were plated per well in a 12 well plate. The next day cells were transduced with 250 uL of supernatant containing TMPRSS2-encoding lentivirus for 16 hr at 37°C, after which fresh media was added to cells. 48 hrs after transduction cells were replated and selected with blasticidin (InvivoGen, catalogue #ant-bl-1) at 10 ug/ml. After selection, cells were transduced similarly with supernatant containing ACE2-encoding lentivirus and selected with 1 ug/mL of puromycin (InvivoGen, San Diego, CA, catalogue #ant-pr-1).
1 × 106 SupT1 cells were transduced with 400 uL of supernatant containing TMPRSS2-encoding virus followed by selection with 10 ug/mL blasticidin 48 hrs later. TMPRSS2 expressing SupT1 cells were then transduced with a second vector expressing ACE2, followed by puromycin selection at 1 ug/mL.
1 × 106 SupT1 cells were transduced with 400 uL of supernatant containing TMPRSS2-encoding virus followed by selection with 10 ug/mL blasticidin 48 hrs later. TMPRSS2 expressing SupT1 cells were then transduced with a second vector expressing ACE2, followed by puromycin selection at 1 ug/mL.
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