The model systems for the cell culture are NCI-H322 (ATCC, Manassas, VA) human bronchioalveolar carcinoma cells, which were maintained in Dulbecco’s modified Eagle’s medium (Corning Cellgrow, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Peak Serum, Fort Collins, CO), 100 U penicillin and 100 mg streptomycin (ThermoFisher, Waltham, MA, USA) and maintained at 37 °C in a humidified atmosphere of 5% CO2. Cells were transfected using Lipofectamine LTX with the addition of the manufacture’s PLUS reagent (Invitrogen, Carlsbad, CA, USA). Cells transfected with pBOS-H2B-GFP were selected with 3 μg/mL Blasticidin S HCl (Sigma-Aldrich, St. Louis, MO, USA), FACS sorted based on positive GFP expression, and maintained in 1 μg/mL Blasticidin S HCl (ThermoFisher, Waltham, MA, USA) within the culture medium. Cells transfected with pCMV-Tag2B-14-3-3γ were maintained in 400 μg/mL G418 (ThermoFisher, Waltham, MA, USA).
Blasticidin s hcl
Manufactured by Merck Group
Sourced in United States
Blasticidin S HCl is a broad-spectrum antibiotic used as a selective agent in cell culture applications. It inhibits protein synthesis by interfering with the peptidyl transferase activity of the 60S ribosomal subunit.
Automatically generated - may contain errors
Lab products found in correlation
Zeocin, by Thermo Fisher Scientific (3 mentions)
Zeocin, by Merck Group (2 mentions)
Yeast nitrogen base, by BD (2 mentions)
Bacto agar, by BD (2 mentions)
Pichia pastoris strain gs115, by Thermo Fisher Scientific (2 mentions)
Bacto tryptone, by BD (2 mentions)
Bacto yeast extract, by BD (2 mentions)
Bacto peptone, by BD (2 mentions)
Carbenicillin, by Duchefa Biochemie (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Nci h322, by American Type Culture Collection (1 mentions)
Blasticidin s hcl, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Hiprep 26 60 sephacryl s 200 hr size exclusion column, by GE Healthcare (1 mentions)
Drosophila schneider 2 cells, by Thermo Fisher Scientific (1 mentions)
Drosophila expression system, by Thermo Fisher Scientific (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Pog44, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
6 protocols using blasticidin s hcl
1
Establishing NCI-H322 Carcinoma Cell Line
2
Drosophila Cell Culture and Protein Purification
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Drosophila Schneider 2 (S2)
cells and Drosophila Expression System were purchased
from Invitrogen. Blasticidin S-HCl was purchased from Sigma-Aldrich.
Insect Xpress growth medium was acquired from Lonza. Anion-exchange
chromatography medium was purchased from Sigma-Aldrich. The HiPrep
26/60 Sephacryl S-200 HR size exclusion column was from GE Healthcare.
Assay reagents were obtained from Sigma-Aldrich or Fisher Scientific.
Methanol (Fisher Optima grade, 99.9%) and purified water (to a resistivity
of 18.2 MΩ·cm at 25 °C using a Milli-Q Gradient ultrapure
water purification system) were used to prepare mobile-phase solvents
for HPLC.
cells and Drosophila Expression System were purchased
from Invitrogen. Blasticidin S-HCl was purchased from Sigma-Aldrich.
Insect Xpress growth medium was acquired from Lonza. Anion-exchange
chromatography medium was purchased from Sigma-Aldrich. The HiPrep
26/60 Sephacryl S-200 HR size exclusion column was from GE Healthcare.
Assay reagents were obtained from Sigma-Aldrich or Fisher Scientific.
Methanol (Fisher Optima grade, 99.9%) and purified water (to a resistivity
of 18.2 MΩ·cm at 25 °C using a Milli-Q Gradient ultrapure
water purification system) were used to prepare mobile-phase solvents
for HPLC.
3
Genetically Modified P. falciparum Lines
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P. falciparum lines 3D7/attB [22 (link)], 3D7/PfPKGT618Q [6 (link)], and 3D7/PfPKG-HA [35 (link)] were cultured according to standard procedures [36 (link)] in human A+ erythrocytes (National Blood Transfusion Service, UK) and RPMI 1640 medium supplemented with 0.5% Albumax type II (Lifetech) and 5 nM WR99210 (Jacobus Pharmaceuticals, New Jersey).
3D7/attB ring stage parasites were co-transfected with the pPfPKG/attP expression plasmid and the bxb1 integrase plasmid (pINT) to facilitate recombination of the attP plasmid with the attB site integrated into the cg6 pseudogene locus as described [22 (link)]. The following drugs were applied 24 hours post transfection for drug selection: G418 (Sigma, 250 μg/ml) for five days to select for the presence of pINT, and blasticidin S HCl (Sigma, 5 μg/ml) to select for the presence of the pPfPKG/attP plasmid. Blasticidin-resistant parasite cultures were established approximately three weeks post transfection. Transfectant cultures were maintained on blasticidin and WR99210 pressure.
Parasite synchronization was achieved by repeated sorbitol treatments. Highly synchronous parasites were obtained by magnet purification of mature schizonts (MACS, Miltenyi Biotech) followed by the addition of fresh erythrocytes and a sorbitol treatment three to five hours later.
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P. falciparum lines 3D7/attB [22 (link)], 3D7/PfPKGT618Q [6 (link)], and 3D7/PfPKG-HA [35 (link)] were cultured according to standard procedures [36 (link)] in human A+ erythrocytes (National Blood Transfusion Service, UK) and RPMI 1640 medium supplemented with 0.5% Albumax type II (Lifetech) and 5 nM WR99210 (Jacobus Pharmaceuticals, New Jersey).
3D7/attB ring stage parasites were co-transfected with the pPfPKG/attP expression plasmid and the bxb1 integrase plasmid (pINT) to facilitate recombination of the attP plasmid with the attB site integrated into the cg6 pseudogene locus as described [22 (link)]. The following drugs were applied 24 hours post transfection for drug selection: G418 (Sigma, 250 μg/ml) for five days to select for the presence of pINT, and blasticidin S HCl (Sigma, 5 μg/ml) to select for the presence of the pPfPKG/attP plasmid. Blasticidin-resistant parasite cultures were established approximately three weeks post transfection. Transfectant cultures were maintained on blasticidin and WR99210 pressure.
Parasite synchronization was achieved by repeated sorbitol treatments. Highly synchronous parasites were obtained by magnet purification of mature schizonts (MACS, Miltenyi Biotech) followed by the addition of fresh erythrocytes and a sorbitol treatment three to five hours later.
4
Bacterial Cultivation and Protein Expression
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Escherichia coli (E. coli) MC1061 or DH5α was used as the host strain for recombinant DNA manipulations. E. coli were cultivated in LB medium supplemented with the appropriate antibiotics: 100 μg/mL carbenicillin (Duchefa Biochemie) or 50 µg/mL Zeocin® (Life Technologies) depending on the required selection.
The Pichia pastoris GS115-strain (Life technologies) was used for protein expression and is the starting strain for glyco-engineering. P. pastoris cells were grown in liquid YPD or solid YPD-agar and selected for with the appropriate antibiotics: 100 µg/mL Zeocin® or 300 µg/mL BlasticidinS-HCl (Sigma Aldrich). Transformants of auxotrophic strains were selected on Complete Supplemented Medium (CSM) lacking Histidine (0.077% CSM-HIS, 1% D-glucose, 1.34% Yeast Nitrogen Base, 1.5% agar). Bacto yeast extract, Bacto tryptone, Bacto peptone, Bacto agar and Yeast Nitrogen Base (YNB) were purchased from Difco (Becton Dickinson). For protein expression, biomass was grown on BMGY. For induction, the cultures were switched to BMMY.
The Pichia pastoris GS115-strain (Life technologies) was used for protein expression and is the starting strain for glyco-engineering. P. pastoris cells were grown in liquid YPD or solid YPD-agar and selected for with the appropriate antibiotics: 100 µg/mL Zeocin® or 300 µg/mL BlasticidinS-HCl (Sigma Aldrich). Transformants of auxotrophic strains were selected on Complete Supplemented Medium (CSM) lacking Histidine (0.077% CSM-HIS, 1% D-glucose, 1.34% Yeast Nitrogen Base, 1.5% agar). Bacto yeast extract, Bacto tryptone, Bacto peptone, Bacto agar and Yeast Nitrogen Base (YNB) were purchased from Difco (Becton Dickinson). For protein expression, biomass was grown on BMGY. For induction, the cultures were switched to BMMY.
5
Flp-In System Generation of Stable Cell Lines
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U2OS Flp-In T-REx, HeLa Flp-In T-REx, or HeLa DR-GFP Flp-In cells were grown in medium supplemented with 100 μg/mL Zeocin (Invitrogen). To generate stable cell lines in the Flp-In system, cells were co-transfected with pOG44 (Invitrogen) and a pcDNA5/FRT plasmid of interest using the Fugene 6 transfection kit (Promega) or Lipofectamine 2000 (Invitrogen). After transfection, Flp-In cells were selected in medium supplemented with 200 μg/mL Hygromycin B (Invitrogen). Individual clones were selected and analyzed for expression. For T-REx cells, selection included 5 μg/mL blasticidin S HCl (Sigma).
6
Bacterial Cultivation and Protein Expression
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Escherichia coli (E. coli) MC1061 or DH5α was used as the host strain for recombinant DNA manipulations. E. coli were cultivated in LB medium supplemented with the appropriate antibiotics: 100 μg/mL carbenicillin (Duchefa Biochemie) or 50 µg/mL Zeocin® (Life Technologies) depending on the required selection.
The Pichia pastoris GS115-strain (Life technologies) was used for protein expression and is the starting strain for glyco-engineering. P. pastoris cells were grown in liquid YPD or solid YPD-agar and selected for with the appropriate antibiotics: 100 µg/mL Zeocin® or 300 µg/mL BlasticidinS-HCl (Sigma Aldrich). Transformants of auxotrophic strains were selected on Complete Supplemented Medium (CSM) lacking Histidine (0.077% CSM-HIS, 1% D-glucose, 1.34% Yeast Nitrogen Base, 1.5% agar). Bacto yeast extract, Bacto tryptone, Bacto peptone, Bacto agar and Yeast Nitrogen Base (YNB) were purchased from Difco (Becton Dickinson). For protein expression, biomass was grown on BMGY. For induction, the cultures were switched to BMMY.
The Pichia pastoris GS115-strain (Life technologies) was used for protein expression and is the starting strain for glyco-engineering. P. pastoris cells were grown in liquid YPD or solid YPD-agar and selected for with the appropriate antibiotics: 100 µg/mL Zeocin® or 300 µg/mL BlasticidinS-HCl (Sigma Aldrich). Transformants of auxotrophic strains were selected on Complete Supplemented Medium (CSM) lacking Histidine (0.077% CSM-HIS, 1% D-glucose, 1.34% Yeast Nitrogen Base, 1.5% agar). Bacto yeast extract, Bacto tryptone, Bacto peptone, Bacto agar and Yeast Nitrogen Base (YNB) were purchased from Difco (Becton Dickinson). For protein expression, biomass was grown on BMGY. For induction, the cultures were switched to BMMY.
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