Lung tissues and swabs from lung parenchyma were used for cultivation of SARS-CoV-2 (Table S3 ). After mechanical disruption samples were frozen (−80 °C) and thawed (37 °C) twice to increase cell lysis and viral release. 2 mL OptiPro SFM medium (Gibco) with 4 mM L-Glutamine (Gibco) and 1% penicillin-streptomycin (10,000 U/mL; Gibco) were added to the samples. After centrifugation (10 min, 1500 rcf) the supernatants were filtered through a 0.45 μm membrane filter (Millipore) and inoculated on Vero CCL-81 cells with OptiPro SFM medium with 4 mM L-Glutamine and 1% penicillin-streptomycin in T25 flasks (ThermoFisher). After 3–4 days incubation at 37 °C and 5% CO2, the whole cells were mechanically detached with cell scrapers and passaged including the supernatant on to new Vero CCL-81 cells growing in T75 flasks (ThermoFisher). After 1 week the cells were harvested and supernatants were stored after centrifugation (10 min, 1500 rcf) at −80 °C. Viral load was determined by qRT-PCR as described below.
Opti pro sfm medium
Manufactured by Thermo Fisher Scientific
Opti-Pro SFM medium is a serum-free, animal component-free, and chemically defined cell culture medium designed for the growth and maintenance of a variety of mammalian cell lines. It is a liquid medium that provides the necessary nutrients and growth factors to support cell growth and proliferation in a controlled and defined environment.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pmaxgfp vector, by Lonza (3 mentions)
0.45 μm membrane filter, by Merck Group (2 mentions)
T25 flask, by Thermo Fisher Scientific (2 mentions)
T75 flask, by Thermo Fisher Scientific (2 mentions)
Freestyle tm max reagent, by Thermo Fisher Scientific (2 mentions)
Nucleocounter nc 250 cell counter, by ChemoMetec (2 mentions)
Advancebio sec column, by Agilent Technologies (2 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Protein g sepharosetm 4 fast flow, by GE Healthcare (1 mentions)
Cho s suspension cells, by Thermo Fisher Scientific (1 mentions)
Anti clumping agent, by Thermo Fisher Scientific (1 mentions)
Nucleocounter nc 200 cell counter, by ChemoMetec (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Pei max, by Polysciences (1 mentions)
12 protocols using opti pro sfm medium
1
Cultivation and Quantification of SARS-CoV-2
2
Formulation and Stability of PEI-DNA and LPF-DNA Films
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PEI-DNA complexes were formed by adding 25 μg of pAAV-LacZ in 400 μL of medium to 100 μg of PEI in 400 μL of medium. Lipofectamine (LPF)-DNA complexes were formed by adding 20 μg of pAAV-LacZ in 250 μL (total volume) of Opti-Pro SFM medium (Gibco, Thermo Fisher Scientific) to 20 μL of LPF in 250 μL (total volume) of Opti-Pro SFM medium. Samples remained at RT for 20 min. At this time, 800-μL solutions containing PEI-DNA complexes were mixed with 200 μL of film formulation for a final DNA concentration of 25 μg/mL. Solutions containing LPF-DNA complexes were mixed with film formulation at a v/v ratio of 1:1 for a final DNA concentration of 20 μg/mL. The resulting solutions were dispensed into 100-μL silicon molds (Bold Maker, Amesbury, MA). All films were dried under aseptic conditions at 20°C. When drying was complete, a subset of films were reconstituted in transfection medium for analysis of loss due to drying. Remaining films were peeled and placed in Ziploc-like particle-free bags (American Cleanstat, Irvine, CA) inside a heat-sealed foil bag (Ted Pella, Redding, CA). For most stability studies summarized here, packaged films were stored in stability chambers (Binder, Tuttlingen, Germany) set at 25°C, 60% RH, or at 4°C, 40%–50% RH. Details of each formulation are summarized in Table S1 .
3
Transient Gene Expression in CHO Cells
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A culture containing 5 × 105 host CHO cells was centrifuged (240 g, 24 °C for 3 min), after which the medium was removed. Then, the cell pellet was suspended in 2 mL of Opti-MEM medium (ThermoFisher Scientific). After removing the supernatant by centrifugation, the remainder was resuspended in 2 mL of Opti-MEM medium, and 1 mL of the culture was dispensed into two wells of a 24-well culture plate. Mixtures of 3.2 µg of expression vectors and 68 µL of Opti-Pro SFM medium (ThermoFisher Scientific) were mixed with a mixture of 8 µL of gene transfer reagents and 68 µL of Opti-Pro SFM medium and reacted at room temperature for 20 min. Half of the reactants were added to each of the dispensed two wells and incubated statically in a CO2 incubator under conditions of 5% CO2 and 37 °C.
4
Cell Line Cultivation and Passaging
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Caco-2 and HT-29 (German collection of microorganisms and cell cultures, ACC 169 and ACC 299, respectively), and HCT-8 (ATCC CCL-244) were cultured in Quantum 286 epithelial medium (GE Healthcare). Vero-B4 cells (ACC-33) were grown in OptiPRO SFM medium (Gibco). The passages used were: Caco-2, 21 to 32; HCT-8, 8 to 21; HT-29, 16 to 20; Vero, 15 to 19.
5
Transient Transfection and Purification of Recombinant Antibodies
6
Overexpression of Therapeutic Proteins in CHO Cells
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Cells were seeded in 250 mL Corning vent cap shake flasks (Sigma-Aldrich) as duplicates with cell densities ~1 x 10 6 cells/mL in 60 mL CD CHO medium supplemented with 8 mM L-glutamine (Life Technologies) and transfected with 75 μg of A1AT-Glul-ST6GAL1 plasmid or 75 μg of C1INH-Glul-ST6GAL1 plasmid (Suppl. Fig. 1) using FreeStyle TM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to the manufacturer's recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection. pmaxGFP ® vector (Lonza) transfection was performed to measure transfection efficiencies. Two days after transfection, cells were transferred into 60 mL CD CHO medium lacking L-glutamine (Life Technologies) and supplemented with 1μL/mL anti-clumping agent and 0 μM, 10 μM, 30 μM or 50 μM MSX (EMD Millipore, Billerica, MA).
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). The cells were passaged in fresh selection medium every 2-3 days until viability and doubling time reached stable values. Polyclonal cell lines (pools) were seeded in duplicates at ~1 x 10 6 cells/mL with corresponding MSX concentrations. Cell densities and viabilities were determined once per day and supernatants of the pools were harvested three days after seeding and pooled within duplicates for purification of rhA1AT and rhC1INH.
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). The cells were passaged in fresh selection medium every 2-3 days until viability and doubling time reached stable values. Polyclonal cell lines (pools) were seeded in duplicates at ~1 x 10 6 cells/mL with corresponding MSX concentrations. Cell densities and viabilities were determined once per day and supernatants of the pools were harvested three days after seeding and pooled within duplicates for purification of rhA1AT and rhC1INH.
7
Transient Expression of Rituximab and EPO
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For transient expression of rituximab/EPO, cells were seeded in Corning vent cap shake flasks (Sigma-Aldrich) as duplicates with cell densities ~1 x 10 6 cells/mL in 60 mL CD CHO medium supplemented with 8 mM L-glutamine (Life Technologies). Cells were incubated in a humidified incubator at 120 rpm, 37 °C and 5% CO2 and transfected with 75 μg of rituximab or EPO encoding plasmid for each flask using FreeStyle TM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection. pmaxGFP ® vector (Lonza) transfection was performed to measure transfection efficiencies.
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). To purify rituximab and EPO, the supernatants of the transfected clones were harvested three days after transfection and pooled within duplicates.
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). To purify rituximab and EPO, the supernatants of the transfected clones were harvested three days after transfection and pooled within duplicates.
8
CRISPR Transfection of CHO-S Cells
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CHO-S suspension cells (Life Technologies, Carlsbad, CA) were cultivated in CD CHO medium supplemented with 8 mM L-glutamine and 1 μL/mL anti-clumping agent (Life Technologies). Cells were incubated in a humidified incubator at 120 rpm, 37 °C and 5% CO2.
Cell passaging was conducted every two to three days at 3 x 10 5 cells/mL after measuring viable cell densities (VCDs) and viabilities with the NucleoCounter NC-200 Cell Counter (ChemoMetec, Allerod, Denmark). One day prior transfection with CRISPR reagents, anti-clumping agent was removed by centrifugation and 5 -6 x 10 5 cells/mL were seeded in a six multi-well well plate (BD Biosciences, San Jose, CA) for each transfection. At the day of transfection each sample was seeded at 1 x 10 6 cells/mL and a total DNA load of 3.5 μg was transfected with FuGENE ® HD transfection reagent (Promega, Madison, WI) and OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. The GFP_2A_Cas9 / sgRNA plasmid ratios for each sample are presented in Table S3. To measure transfection efficiency, pmaxGFP ® vector (Lonza, Basel, Switzerland) transfection was performed. Cells were harvested for fluorescence-activated cell sorting (FACS) 48 h after transfection.
Cell passaging was conducted every two to three days at 3 x 10 5 cells/mL after measuring viable cell densities (VCDs) and viabilities with the NucleoCounter NC-200 Cell Counter (ChemoMetec, Allerod, Denmark). One day prior transfection with CRISPR reagents, anti-clumping agent was removed by centrifugation and 5 -6 x 10 5 cells/mL were seeded in a six multi-well well plate (BD Biosciences, San Jose, CA) for each transfection. At the day of transfection each sample was seeded at 1 x 10 6 cells/mL and a total DNA load of 3.5 μg was transfected with FuGENE ® HD transfection reagent (Promega, Madison, WI) and OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. The GFP_2A_Cas9 / sgRNA plasmid ratios for each sample are presented in Table S3. To measure transfection efficiency, pmaxGFP ® vector (Lonza, Basel, Switzerland) transfection was performed. Cells were harvested for fluorescence-activated cell sorting (FACS) 48 h after transfection.
9
Trastuzumab Production with Ncaa Incorporation
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For each transfection 20 mL of FreeStyle™ 293-F cells were prepared at a cell density of 1 × 106 cells/mL. 20 µg DNA were used for each transfection using four different plasmids in equal amounts: (1) trastuzumab LC plasmid, (2) trastuzumab HC121TAG or trastuzumab HC plasmid, (3) NES-PylRSAF + tRNAPyl plasmid, and (4) mock plasmid (pcDNA3.1_Zeo+) or pl.18-HA-PKR-DN (PKR∆). The plasmids were pipetted into 2 mL OptiPRO™ SFM medium (Thermo Fisher Scientific, 12309019), mixed with 80 µL PEI MAX (Polysciences, 24765-100, 1 mg/mL stock prepared following manufactures instruction), vortexed 3 × 5 s and incubated for 10 min at RT before added into the cell suspension. The expression was incubated for 6 days at 37 °C, 8% CO2 at 120 rpm (50 mm shaking throw). Samples were incubated in the presence of 500 μM cyclooctyne-lysine (SCO-K, SiChem, SC-8000) in the medium buffered with 25 mM HEPES (pH 7.25).
10
Xyloside treatment of Actin-EmGFP A549 cells
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Actin-EmGFP-modified A549 cells were cultured to approx. 70% confluence in DMEM/F-12/GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 100 units per mL penicillin and 100 μg mL−1 streptomycin (Sigma-Aldrich). For xyloside treatment, the growth medium was aspirated and xylosides (100 μM from 50 mM stock solution in DMSO) in OptiPRO SFM medium (Thermo Fisher Scientific) was added to the cells. After 24 h of treatment, medium samples were either analyzed directly by fluorescence detection size-exclusion chromatography (FSEC) on an AdvanceBio SEC column (Agilent) or purified on an anion exchange DE52 diethylaminoethyl cellulose resin (Sigma-Aldrich). Purified samples were subsequently analyzed by FSEC or reversed-phase chromatography on an ACE C8, 3 μm 4.6 × 100 mm (ACE).
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