HFFs were subjected to lentiviral transduction and selected for stable expression of pUL97. To this end, replication-deficient lentiviruses were prepared by cotransfection of 293T cells with a pLenti6/V5-D-TOPO vector coding for pUL97 together with a packing plasmid mix coding for HIV-1 Gag/Pol, HIV-1 Rev and the envelope protein G of vesicular stomatitis virus (expression vectors pLP1, pLP2, and pVSV-G, respectively) using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany). Viral supernatants were harvested 48 h post-transfection, subjected to centrifugation to remove debris, filtered, and stored in aliquots at −80 °C. HFFs of a low passage number were incubated for 24 h with various amounts of lentiviral supernatant in the presence of 7.5 μg·mL−1 polybrene (Sigma-Aldrich, Taufkirchen, Germany). Next, virus supernatants were replaced by fresh media and cells were incubated for another 24 h before blasticidin (2 μg·mL−1, PAA Laboratories, Cölbe, Germany) was added in order to select for stably transduced cell populations.
Blasticidin
Manufactured by GE Healthcare
Sourced in Germany
Blasticidin is a broad-spectrum antibiotic that inhibits protein synthesis in eukaryotic cells. It is commonly used as a selectable marker in cell culture and genetic engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Nocodazole, by Merck Group (2 mentions)
Zeocin, by InvivoGen (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pog44, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Reversine, by Merck Group (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Ultraglutamine, by Lonza (1 mentions)
Hygromycin b, by Roche (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Puromycin, by GE Healthcare (1 mentions)
6 protocols using blasticidin
1
Lentiviral Transduction of HFFs for pUL97 Expression
2
Inducible Protein Expression in HeLa Flp-in Cells
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HeLa Flp-in cells (gift from S. Taylor, University of Manchester, England, UK) stably expressing a TetR, were cultured in DMEM (4.5 g/L glucose, Lonza) supplemented with 9 % fetal bovine serum (Tetracyclin-approved, Lonza), 50 μg/ml penicillin/streptomycin (Gibco), and 2 mM Ultraglutamine (Lonza). All HeLa Flp-in cell lines stably carrying doxycycline-inducible eYFP-FKBP-MAD1 constructs were transfected with pcDNA5/FRT/TO (Invitrogen) and pOG44 (Invitrogen) plasmid-carrying Flp-recombinase. Selection and maintenance of stable cells was done in medium supplemented with 200 μg/ml Hygromycin B (Roche) and 4 μg/ml blasticidin (PAA Laboratories). HeLa Flp-in cell lines stably expressing MIS12-FRB constructs were transfected with Fugene HD (Roche), and stable lines were selected for using 2 μg/ml puromycin (Sigma). The HeLa Flp-in cell lines expressing cyclin B1-mCherry were transfected with pcDNA3-cyclin B1-mCherry and, stable cell lines were selected using 100 μg/ml Zeocin (Invivogen). The reagents thymidine (2 mM), reversine (500 nM), nocodazole (830 μM), MG132 (10 μM), and doxycycline (1 μg/ml) were purchased from Sigma-Aldrich and used at final concentrations indicated. Rapamycin (100 nM) was purchased from LC-Laboratories.
3
Drosophila and Cell Culture Maintenance
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Drosophila D.mel-2 (Invitrogen) and C3 cells were maintained in Express Five® SFM media (Invitrogen,) supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Sigma-Aldrich) at 25°C in an cooled incubator. Expression of the reporter construct in C3 cells was maintained by the addition of 5μg/mL Blasticidin (PAA Laboratories). HeLa-M and C1 cells were grown in high glucose DMEM supplemented with 10% fetal calf serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Sigma-Aldrich) at 37°C in a 5% CO2 humidified incubator. Expression of the reporter construct in C1 cells was maintained by the addition of 1.66μg/mL puromycin (PAA Laboratories). siRNA transfections were performed as in Gordon et al., 2010. The sequence of the siRNA used in the experiments can be found in (S2 Table ).
4
Cell Line Cultivation and CPAF Induction
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All cell lines were cultured at 37°C and with5% CO2. HeLa cells and 293-TRex-3xGyrB-CPAF cells (16 (link)) were maintained in Dulbecco modified Eagle’s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) (tetracycline negative; PAA Laboratories) or 10% fetal bovine serum (FBS) (Gibco). For the 293-TRex-3xGyrB-CPAF cells, 5 µg/ml blasticidin (PAA Laboratories) and 350 µg/ml zeocin (Invivogen) were added. To induce and activate CPAF, 5 ng/ml anhydrotetracycline (AHT) (IBA Life Sciences) and 1 µM coumermycin (CM) (Sigma) were added (16 (link)). For some experiments, cytochalasin D (Sigma), nocodazole (Sigma), or blebbistatin (Calbiochem/ EMD Millipore) was added.
5
HeLa Cell Culture Protocol
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HeLa cells and HeLa Flp-In T-Rex cells were cultured in DMEM (Sigma-Aldrich) supplemented with 6% FCS (FBS; Sigma-Aldrich), 2 mM UltraGlutamine, and 100 U/ml penicillin and 100 µg/ml streptomycin (Lonza). Culture medium of HeLa Flp-In T-Rex cells and of all HeLa Flp-In T-Rex–derived cell lines (described below) was additionally supplemented with 4 µg/ml Blasticidin (PAA Laboratories). All cell lines were cultured at 37°C with 5% CO2.
6
Generating C3 Cell Line with Reporter Construct
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The reporter construct used to generate the C3 cell line was generated by subcloning the expression cassette from pC4S1-eGFP-FM4-FCS-hGH (Ariad Pharmaceuticals) into pAC-V5-His-A expression vector (Invitrogen). 2μg of pAC-S1-eGFP-FM4-FCS-hGH was co-transfected with 50ng of pCoBLAST into 500,000 S2 cells using the TransFast transfection reagent (Promega). A population of cells stably expressing the reporter construct was generated by the addition of 25 μg/mL blasticidin (PAA Laboratories). The cells were selected for two weeks and then autocloned into a 96 well plate using a MoFlo Flow cytometer (Beckman Coulter) based on GFP fluorescence. We were initially unsuccessful in this process until we supplemented the media with 5% FCS and put two cells in each well of the plate. 96 well plates were sealed with Parafilm M (Pechiney Plastic Packaging) to minimize evaporation during cell culture. Positive wells were identified using fluorescence microscopy. Clonal cell lines were screened for their ability to efficiently secrete the reporter construct and Clone 3 cells chosen as they have the most uniform expression of the reporter construct.
HA tagged Drosophila STX1 was generated using PCR and cloned into the copper inducible expression vector pMT/V5-HIS (Invitrogen). A stable population of cells was generated by co-transfecting the plasmid with pCoBLAST and selected as above.
HA tagged Drosophila STX1 was generated using PCR and cloned into the copper inducible expression vector pMT/V5-HIS (Invitrogen). A stable population of cells was generated by co-transfecting the plasmid with pCoBLAST and selected as above.
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